AU2017201593B2 - Use of anatabine to treat inflammation and methods of synthesizing anatabine - Google Patents

Abstract

H:\xd\Interwoven\NRPortbl\DCC\SXD\I33562121.docx-8/03/2017 Pharmaceutical compositions comprising an isolated form of anatabine or a salt thereof can be used to treat disorders comprising an inflammatory component, including chronic, low level inflammation. In another aspect, methods for the synthetic preparation of anatabine involve reacting benzophenoneimine with 3-aminomethyl pyridine to form benzylhydrylidene pyridin-3-yl-methyl-amine. The benzylhydrylidene-pyridin-3-yl-methyl-amine is treated with a non-nucleophilic base and a dielectrophile, such as cis- 1,4-dichloro-2-butene, followed by acidification, then basification, to provide anatabine. 100 ______ E_ RACEMIO MIXTURE )S (S-(-)-ANATABINE Lu co40 0 10 100 200 400 600 800 1000 DOSE (pg/mL)

Description

BACKGROUND [02] Inflammation is a protective response to harmful stimuli, such as oxidative stress, irritants, pathogens, and damaged cells. The inflammatory response involves the production and release of inflammatory modulators that heal injured tissue and destroy damaged cells, by directly or indirectly producing and/or signaling the release of agents that produce reactive oxygen species. Thus, an appropriate inflammatory response involves a balance between the destruction of damaged cells and the healing of injured tissue.

[03] An unchecked inflammatory response can lead to oxidative stress and the onset of various inflammatory disease pathologies. In fact, inflammatory processes underlie a wide variety of pathologies, including immune and autoimmune diseases, gastrointestinal diseases, various types of cancer, vascular disorders, heart disease, and neurodegenerative diseases. There is a need in the art for agents that can reduce inappropriate levels of inflammation.

SUMMARY [04] A pharmaceutical composition comprising a therapeutically effective dose of an isolated form of anatabine or a pharmaceutically acceptable salt thereof can be administered to an individual to reduce a symptom of a disorder comprising an NFkBmediated inflammatory component or to reduce the risk of developing such a disorder.

2017201593 08 Mar 2017 [05] In some embodiments the NFicB-rnediated inflammatory component is chronic inflammation which occurs, for example, in thyroiditis, cancer, arthritis, Alzheimer’s disease, and multiple sclerosis.

[06] In other embodiments, a therapeutically effective dose of an isolated form of anatabine or a pharmaceutically acceptable salt thereof can be administered to an individual for treating alopecia areata or other disorders associated with hair loss.

[07] In some embodiments therapeutically effective doses of anatabine are provided in an extended release formulation. In other embodiments, isolated forms of anatabine or pharmaceutically effective salts thereof can be provided in a bottled water product comprising about 100 ml to about 2,000 ml purified water and from about 0.00001 to about 0.0001 wt% of anatabine or a water-soluble salt of anatabine.

[08] In another aspect, a method is provided to prepare anatabine synthetically. In a first step, benzylhydrylidene-pyridin-3-ylmethyl-amine (Formula I) is prepared by reacting 3-aminomethylpyridine with benzophenoneimine, or benzophenoneimine substituted in one or both aromatic rings.

Ph

Formula I |09| The compound of Formula I may then be reacted with a non-nucleophilic base and a dielectrophile to obtain a compound of Formula II.

[ίο] [10]

Formula II

The compound of Formula II is then acidified to provide a compound of Formula III.

2017201593 08 Mar 2017 [ill [12] [13] [14] [11] [12] [13] [14]

Formula III

The compound of Formula III is basified to yield anatabine.

In another aspect, a novel method is provided to recover anatabine from a reaction product by contacting the reaction product with methyl t-butyl ether (MTBE). Subsequence distillation yields anatabine with a purity greater than 99%.

In yet another aspect, acceptable pharmaceutical or food-grade salts of anatabine are prepared.

In an alternative embodiment, methods are provided for synthesizing other minor alkaloids, such as anabasine, nornicotine, N-methylanabasine, or anabaseine. A compound of Formula I may be prepared as previously described and reacted with a dielectrophile to yield a compound of Formula Ila:

Ph'

Ph

Formula Ila

115] The dielectrophile may be selected according to the desired alkaloid. The compound of Formula Ila is then acidified to provide a compound of Formula Illa.

2017201593 08 Mar 2017

116] Basification of the compound of Formula Illa results in the desired alkaloid by intramolecular N-alkylation.

BRIEF DESCRIPTION OF THE DRAWINGS

117] FIG. 1. Graph showing effects of anatabine (AN) on TNFa-induced NFkB activity in vitro. See Example 1.

[18] FIG. 2. Graph showing effects of a crude extract of smokeless tobacco on TNFainduced NFkB activity in vitro. See Example 1.

[19] FIG. 3. Graph showing effects of nicotine and of an alkaloid extract of smokeless tobacco on TNFa-induced NFkB activity in vitro. See Example 1.

[20] FIG. 4. Graph showing the results of a cytotoxicity assay measuring release of lactate dehydrogenase (LDH) using supernatant from the cells assayed in FIG. 1. See Example 2.

[21] FIG. 5. Graph showing the results of a cytotoxicity assay using supernatant from the cells assayed in FIG. 2. See Example 2.

[22] FIG. 6. Graph showing the results of a cytotoxicity assay using supernatant from the cells assayed in FIG. 3. See Example 2.

2017201593 08 Mar 2017 [23] FIG. 7. Graph showing concentrations in rat plasma as a function of time of anatabine and nicotine after a single intravenous bolus injection.

[24] FIG. 8. Graph showing concentrations of anatabine and nicotine in rat plasma as a function of time (semi-log).

[25] FIG. 9. Graph showing AUCo-»® versus dose for both anatabine and nicotine in male and female rats.

[26] FIG. 10. Graph showing concentrations of anatabine or nicotine in rat brain extracts following a single intravenous bolus dose.

[27] FIG. 11. Graph showing mean concentration of anatabine and nicotine in rat brain extracts 0.5 hours after a single intravenous bolus dose.

[28] FIG. 12. Nicotine product ion scan.

[29] FIG. 13. Nicotine sample chromatogram.

[30] FIG. 14. Anatabine product ion scan.

[31] FIG. 15. Anatabine sample chromatogram.

[32] FIG. 16. Nicotine-d3 product ion scan.

[33] FIG. 17. Nicotine-d3 sample chromatogram.

[34] FIG. 18. Anatabine-d4 product ion scan.

[35] FIG. 19. Anatabine-d4 sample chromatogram.

[36] FIG. 20. Graph showing mean body weights (± Std Dev) for each treatment group and gender.

[37| FIGS. 21A-21B. Graphs showing mean (± SEM) concentration of anatabine in plasma for male or female rats. FIG. 21A, 0.6 mg/kg body weight (BW); FIG. 21B, 6.0 mg/kg BW.

2017201593 08 Mar 2017 [38] FIGS. 22A-22B. Graphs showing mean (± SEM) concentration of anatabine in plasma for male and female rats combined. FIG. 22A, 0.6 mg/kg BW; FIG. 22B, 6.0 mg/kg BW.

[39] FIGS. 23A-23B. Graphs showing mean (± SEM), maximal (Cp, max), and minimal (Cp, min) concentrations of anatabine in plasma for male or female rats. FIG. 23A, 0.6 mg/kg BW; FIG. 23B, 6.0 mg/kg BW.

[40] FIG. 24. Graph showing effect of S-(-)-anatabine on TNFa-induced NFkB activity in vitro.

DETAILED DESCRIPTION [41] This disclosure describes methods of using a composition comprising an isolated form of anatabine or a salt thereof to treat disorders comprising an inflammatory component, including chronic, low-level inflammation. Anatabine is an alkaloid present in tobacco and, in lower concentrations, in a variety of foods, including green tomatoes, green potatoes, ripe red peppers, tomatillos, and sundried tomatoes. Preparation of isolated forms of anatabine is described below. Use of such isolated forms avoids the toxicity associated with tobacco, tobacco extracts, alkaloid extracts, and nicotine.

[42] Without being bound by this explanation, data presented in Examples 1 and 2 below indicate that anatabine reduces transcription mediated by nuclear factor kB (NFkB). NFkB is a transcription factor which operates in cells involved in inflammatory and immune reactions. As documented in Table 1, NFicB-mediated transcription is associated with numerous disorders, including those with an inflammatory component, an aberrant immune response, and/or inappropriate cell proliferation. Isolated forms of anatabine or salts of such isolated forms anatabine are particularly useful for treating disorders comprising an “NFicB-mediated inflammatory component,” i.e. inflammation characterized by, caused by, resulting from, or affected by NFicB-mediated transcription.

2017201593 08 Mar 2017 [43] In other embodiments, a therapeutically effective dose of an isolated form of anatabine or a pharmaceutically acceptable salt thereof can be administered to an individual for treating alopecia areata or other disorders associated with hair loss.

[44] “Treat” as used herein refers to reducing a symptom of the inflammation or resulting disorder but does not require complete cure, either of the inflammation or the disorder. “Reduction of a symptom” of a disorder with an NFKB-mediated inflammatory component includes but is not limited to elimination of the symptom as well as reduction in frequency, severity, or duration of the symptom. Reduction of a symptom can be recognized subjectively by the individual or an observer of the individual or can be confirmed by clinical and/or laboratory findings.

[45] An isolated form of anatabine or a pharmaceutically acceptable salt of such an isolated form of anatabine is administered to the individual at a dose sufficient to reduce a symptom of a disorder with an NFKB-mediated-transcription component. “Individual” as used herein includes warm-blooded animals, typically mammals, including humans and other primates. Doses typically range from about 1 pg/kg to about 7 mg/kg body weight (e.g., about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,

2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,

4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 pg/kg or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,

0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,

2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4-.5, 4.6,

4.7, 4.8, 4.9, or 5 mg/kg), about 1.5 pg/kg to about 5 pg/kg, about 1 pg/kg to about 10 pg/kg, about 0.01 mg/kg to about 7 mg/kg body weight, about 0.1 mg/kg to about 5 mg/kg; about 0.1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 3 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 1 mg/kg to about 2 mg/kg, about 3 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 5 mg/kg, or about 0.5 mg/kg to about 1.5 mg/kg. Certain factors may influence the dose sufficient to reduce a symptom of a disorder (i.e., an effective dose), including the severity of the disease or disorder, previous treatments, the general health, age, and/or weight of the individual, the frequency of treatments, the rate of release from the composition, and other diseases present. This dose may vary according to factors such as the disease state, age, and weight of the subject. For example, higher doses may be administered for treatments involving conditions which are at an advanced stage and/or life-

2017201593 08 Mar 2017 threatening. Dosage regimens also may be adjusted to provide the optimum therapeutic response.

[46] In some embodiments tablets comprising about 600 pg anatabine citrate are administered from once to 25 times daily (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25) times daily.

[47] In some embodiments the dose sufficient to reduce the symptom of the disorder can include a series of treatments. For example, an individual can be treated with a dose of an isolated form of anatabine or a salt thereof several times per day (e.g., 2-12 or 410 times per day), once daily, or less frequently such as 1-6 times per week. Treatments may span between about 1 to 10 weeks (e.g., between 2 to 8 weeks, between 3 to 7 weeks, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks). It will also be appreciated that a dose regimen used for treatment may increase or decrease over the course of a particular treatment.

]48] In some embodiments the individual is an animal, such as a companion animal, a service animal, a farm animal, or a zoo animal. Such animals include, but are not limited to, canines (including dogs, wolves), felines (including domestic cats, tigers, lions), ferrets, rabbits, rodents (e.g., rats, mice), guinea pigs, hamsters, gerbils, horses, cows, pigs, sheep, goats, giraffes, and elephants.

[49] In some embodiments an isolated form of anatabine or a pharmaceutically acceptable salt of an isolated form of anatabine can be administered to reduce the risk of developing a disorder comprising an NFxB-mediated inflammatory component (i.e., prophylactically). One can readily identify individuals with an increased risk or family history of a disorder. Other recognized indices of elevated risk of certain disorders can be determined by standard clinical tests or medical history.

|50] NFKB-mediated transcription is implicated in an enormous variety of maladies. Based on S-(-)-anatabine’s surprising efficacy in interfering with or interrupting this pivotal inflammatory-related activity, anatabine can be expected to have a wide range of therapeutic utilities.

2017201593 08 Mar 2017 [51] In some embodiments the disorder is thyroiditis. In some embodiments the disorder is an immune or autoimmune disorder. In some embodiments the disorder is arthritis, such as rheumatoid arthritis, primary and secondary osteoarthritis (also known as degenerative joint disease). In some embodiments the disorder is a spondyloarthropathy, such as psoriatic arthritis, juvenile chronic arthritis with late pannus onset, and enterogenic spondyloarthropathies such as enterogen ic reactive arthritis, urogenital spondyloarthropathy, and undifferentiated spondylarthropathy. In some embodiments the disorder is a myopathy, such as “soft tissue rheumatism” (e.g., tennis elbow, frozen shoulder, carpal tunnel syndrome, plantar fasciitis, and Achilles tendonitis).

[52] In some embodiments the disorder is diabetes, either type I diabetes or type II diabetes. In other embodiments the disorder is a gastrointestinal inflammatory disorder, such as an inflammatory bowel disease. Examples of inflammatory bowel disease include, but are not limited to, Crohn’s disease, Barrett’s syndrome, ileitis, irritable bowel syndrome, irritable colon syndrome, ulcerative colitis, pseudomembranous colitis, hemorrhagic colitis, hemolytic-uremic syndrome colitis, collagenous colitis, ischemic colitis, radiation colitis, drug and chemically induced colitis, diversion colitis, colitis in conditions such as chronic granulomatous disease, celiac disease, celiac sprue, food allergies, gastritis, infectious gastritis, enterocolitis (e.g., Helicobacter pylori-infected chronic active gastritis), and pouchitis.

[53] In other embodiments the disorder is graft-versus-host-disease (GVHD), systemic lupus erythematosus (SLE), lupus nephritis, Addison’s disease, Myasthenia gravis, vasculitis (e.g., Wegener’s granulomatosis), autoimmune hepatitis, osteoporosis, and some types of infertility.

[54] In some embodiments the disorder is vascular inflammatory disease, associated vascular pathologies, atherosclerosis, angiopathy, inflammation-induced atherosclerotic or thromboembolic macroangiopathy, coronary artery disease, cerebrovascular disease, peripheral vascular disease, cardiovascular circulatory disease such as as ischemia/reperfusion, peripheral vascular disease, restenosis following angioplasty, inflammatory aortic aneurysm, vasculitis, stroke, spinal cord injury, congestive heart failure, hemorrhagic shock, ischemic heart

2017201593 08 Mar 2017 disease/reperfusion injury, vasospasm following subarachnoid hemorrhage, vasospasm following cerebrovascular accident, pleuritis, pericarditis, inflammationinduced myocarditis, or a cardiovascular complication of diabetes.

[55] In some embodiments the disorder is brain swelling or a neurodegenerative disease such as multiple sclerosis, Alzheimer’s disease, or Parkinson’s disease. In other embodiments the disorder is inflammation related to a kidney disease, nephritis, glomerulonephritis, dialysis, peritoneal dialysis, pericarditis, chronic prostatitis, vasculitis, gout, or pancreatitis.

[56] In some embodiments the disorder is an anemia. In other embodiments the disorder is an ulcer-related disease, such as peptic ulcer disease, acute pancreatitis, or aphthous ulcer. In other embodiments the disorder is related to an age-related disease, such as atherosclerosis, fibrosis, and osteoporosis, or a disorder associated with pre-maturity, such as retinopathy, chronic lung disease, arthritis, and digestive problems.

[57] In other embodiments the disorder is preeclampsia, inflammation related to chemical or thermal trauma due to bums, acid, and alkali, chemical poisoning (MPTP/concavalin/chemical agent/pesticide poisoning), snake, spider, or other insect bites, adverse effects from drug therapy (including adverse effects from amphotericin B treatment), adverse effects from immunosuppressive therapy (e.g., interleukin-2 treatment), adverse effects from OKT3 treatment, adverse effects from GM-CSF treatment, adverse effects of cyclosporine treatment, and adverse effects of aminoglycoside treatment, stomatitis and mucositis due to immunosuppression, or exposure to ionizing radiation, such as solar ultraviolet exposure, nuclear power plant or bomb exposure, or radiation therapy exposure, such as for therapy for cancer.

[58] In some embodiments the disorder is a cancer, such as acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, grade I (anaplastic) astrocytoma, grade II astrocytoma, grade III astrocytoma, grade IV astrocytoma, atypical teratoid/rhabdoid tumor of the central nervous system, basal cell carcinoma, bladder cancer, breast cancer, breast sarcoma, bronchial cancer, bronchoalveolar carcinoma, Burkitt lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endometrial

2017201593 08 Mar 2017 cancer, endometrial uterine cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular cancer, Hilar cholangiocarcinoma, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, Langerhans cell histiocytosis, large-cell undifferentiated lung carcinoma, laryngeal cancer, lip cancer, lung adenocarcinoma, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, endocrine neoplasia, multiple myeloma, mycosis fungoides, myelodysplasia, myelodysplastic/ myeloproliferative neoplasms, myeloproliferative disorders, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian clear cell carcinoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, respiratory tract cancer with chromosome 15 changes, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous non-small cell lung cancer, squamous neck cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, cancer of the renal pelvis, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wilms tumor.

[59] In other embodiments, disorders associated with inflammation in the stomach and/or esophagus, such as acid reflux, may be treated.

[60] Anatabine may have a positive effect on telomere length. Thus, in some embodiments anatabine can be administered to reduce and/or reverse cellular senescence (i.e.,

2017201593 08 Mar 2017 aging). Conditions associated with cellular senescence can be treated. For example, anatabine can be administered to reduce and/or reverse senescence of:

a. cells with replicative capacity in the central nervous system, including astrocytes, endothelial cells, and fibroblasts which play a role in age-related diseases as Alzheimer's disease, Parkinson's disease, Huntington's disease, and stroke;

b. cells with finite replicative capacity in the integument, including fibroblasts, sebaceous gland cells, melanocytes, keratinocytes, Langerhan's cells, and hair follicle cells, which may play a role in age-related diseases of the integument such as dermal atrophy, elastolysis and skin wrinkling, sebaceous gland hyperplasia, senile lentigo, graying of hair and hair loss, chronic skin ulcers, and age-related impairment of wound healing;

c. cells .with finite replicative capacity in the articular cartilage, such as chondrocytes and lacunal and synovial fibroblasts, which play a role in degenerative joint disease;

d. cells with finite replicative capacity in the bone, such as osteoblasts and osteoprogenitor cells, which play a role in osteoporosis;

e. cells with finite replicative capacity in the immune system such as B and T lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK cells and their respective progenitors, which may play a role in age-related immune system impairment;

f. cells with a finite replicative capacity in the vascular system including endothelial cells, smooth muscle cells, and adventitial fibroblasts, which may play a role in age-related diseases of the vascular system including atherosclerosis, calcification, thrombosis, and aneurysms;

g. cells with a finite replicative capacity in the eye such as pigmented epithelium and vascular endothelial cells which may play an important role in age-related macular degeneration; and

h. cells with abnormal poliferative capacity, such as cancer cells.

2017201593 08 Mar 2017 [61[ [611 [62] [62] [63] [63]

Isolated forms of anatabine and pharmaceutically acceptable salts of such isolated forms of anatabine can be used in conjunction with (i.e., before, after, or at the same time as) other therapies for any disorder with an NFicB-mediated component. In some embodiments, these therapies include other products that inhibit production of NFkBmediated inflammatory species. These products include, but are not limited to, dexamethasone, glucocorticoids (eg., prednisone, methyl prednisolone), cyclosporine, tacrolimus, deoxyspergualin, non-steroidal antiinflammatory drugs (NSAIDs) such as aspirin and other salicylates, tepoxalin, synthetic peptide proteosome inhibitors, antioxidants (e.g., N-acetyl-L-cysteine, vitamin C, vitamin E, dithiocarbamate derivatives, curcumin), IL-10, nitric oxide, cAMP, gold-containing compounds, and gliotoxin.

Isolated forms of anatabine and pharmaceutically acceptable salts of such isolated forms of anatabine can also be used to improve erectile dysfunction, either administered alone of in conjunction with other therapies such as tadalafil (e.g. CIALIS®), vardenafil (e.g., LEVITRA®, STAXYN®), and sildenafil (e.g., VIAGRA®).

Anatabine

Anatabine can be prepared synthetically. Such synthetic preparation techniques produce isolated forms of anatabine, either as a racemic mixture or as isolated enantiomers, S-(-)-anatabine or R-(+)-anatabine. The chemical structure of anatabine (l,2,3,6-tetrahydro-[2,3']bipyridinyl) is illustrated below, in which * designates an asymmetric carbon.

[64] In one aspect of the invention, methods are provided for synthesizing anatabine, especially methods that are useful in larger scale syntheses. In Step 1, 3-aminomethyl pyridine is reacted with benzophenoneimine (or benzophenoneimine substituted in either or both rings with nitrogen, a halogen, or an alkyl group) to form benzylhydrylidene-pyridin-3-ylmethyl-amine, also referred to herein as Formula I:

2017201593 08 Mar 2017

3-aminomethyl pyridine

benzophenoneimine

Step 1

------►

[65] Step 1 may be performed at any suitable temperature range. The reaction is exothermic, normally resulting in a temperature increase from ambient to about 45 °C to 55 °C. If desired, the reaction mixture may be heated or cooled. In general, the rate of reaction increases at higher temperature and decreases at lower temperatures. For example, step 1 may be performed at about 30 °C to about 60 °C. Most often, step 1 is performed at a temperature of about 45 °C to about 55 °C.

[66] The amount of time needed to complete Step 1 may vary depending on such factors such as reaction temperature and pressure. Normally, the reaction is completed in about 4 to about 9 hours, more usually from about 5 to about 7 hours.

[67] Step 1 is particularly advantageous as it can be scaled up to facilitate larger scale production. For example, the amount of starting material (e.g. combined weight of 3aminomethylpyridine and benzophenoneimine) used for a batch may be about 500 mg, about 1 g, about 5 g, about 10 g, about 20 g, about 25 g, about 30 g, about 40 g, about 50 g, about 100 g, about 200 g, about 500 g, about 1 kg, about 5 kg, or about 10 kg. Often, a medium scale synthesis starts with 20 g to 30 g of starting material. A large scale synthesis may start with 1 kg to 5 kg or more of starting material.

[68] Using benzophenoneimine provides several key advantages over earlier methods and facilitates larger-scale synthesis as described above. First, the reaction by-product is ammonia rather than water. The ammonia escapes from the reaction mixture. By avoiding the presence of water as byproduct, the reaction temperature—50 °C, for

2017201593 08 Mar 2017 example—is much lower than the ~85 °C used for the water-removing benzene reflux step in the method of Deo et al., “Regioselective alkylation of N(diphenylmethylidine)-3-(aminomethyl) pyridine: A simple route to minor tobacco alkaloids and related compounds,” Tetrahedron Letters, Vol. 37, Issue 8, 19 Feb. 1996, pp. 1137-1140. Using lower temperatures in the preparation of the compound offers improved economic and environmental efficiencies. Also, by omitting benzene from the reaction, the synthesis is safer and more environmentally-friendly. Avoiding the presence of water is also beneficial in terms of improved product stability because anatabine is moisture-sensitive.

[69] Second, because benzophenoneimine is liquid, Step 1 may proceed in a solvent-less reaction medium such that the purity of the isolated intermediate is enhanced and avoids the need for a workup reaction. Purity of Formula I compound typically is about 93-95% whereas the Deo et al. method yields only about 83-85% purity of the intermediate.

[70] The purity of Formula I compound may be as high as about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, or about 93%. Often, the Formula I compound is about 93 % to about 95% pure.

[71] In step 2i, the compound of Formula I is reacted with a non-nucleophilic base, such as lithium diisopropylamide (LDA), and a dielectrophile, such as cis-l,4-dichloro-2butene to form a compound of Formula II. The non-nucleophilic base typically is added in a molar excess, for example, at about 1.2 equivalents (eq), about 1.3 eq, about 1.4 eq, about 1.5 eq, about 1.6 eq, about 1.7 eq, about 1.8 eq, about 1.9 eq, or about 2.0 eq. Often the non-nucleophilic base is added in an amount of about 1.3 eq to

2017201593 08 Mar 2017 [72] The non-nucleophilic base is typically supplied in a chilled state to provide improved reagent stability. For example, LDA may be supplied at a temperature of about -30 °C, about -20 °C, about -10 °C, about -5 °C, about 0 °C, about 5 °C, or about 10 °C. Most often the non-nucleophilic base is provided at a temperature of about -30 °C to about 0 °C.

[73] The Step 2i reaction may be incubated for a time suitable for the reaction to go to completion or substantial completion. For example, the reaction time for Step 2i often is about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 1 hour. Often, the reaction is incubated for about 20 to about 40 minutes.

[74] As an alternative to LDA in step 2i, other strong bases such potassium tert butoxide (K^lOBu) may be used. Potassium tert butoxide (K^lOBu) was found to provide improved yield and purity. Using K^lOBu we obtained an anatabine yield of 25% with 97% purity. Other alternatives to LDA include sodium hydride, sodamide, and other alkyllithium reagents.

[75] Following addition of the non-nucleophilic base, a dielectrophile of Formula IV is added:

Formula IV wherein each of Rj and R₂ is independently selected from the group consisting of chlorine, (Cl); bromine, (Br), and iodine, (I), tosylate, mesylate, and triflate. For example, Ri and R2 may both be Cl, such that the dielectrophile is cis-l,4-dichloro-2butene. The dielectrophile may be added neat or in a suitable solvent, such as THF.

[76] For step 2ii, Formula II compound is hydrolyzed with acid. Treatment with acid may be for any suitable time. For example, HCI (10%) treatment may be for about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, or for about 1 hour. Often, acid treatment is for about 10 minutes to about 20 minutes. Other suitable acids include dilute sulfuric acid and phosphoric acid. For step 2iii, Formula III compound is basified, such as by treatment with solid K2CO3, then aqueous KOH solution (e.g., 40%) to achieve N-alkylation to yield anatabine. Other bases, such as

Diaza(l,3)bicyclo[5.4.0]undecane (DBU), di-isopropyl ethylamine, or triethylamine, may also be used.

2017201593 08 Mar 2017

Step 2-ii

------►

Formula II

[77] In another aspect, a method of recovering and purifying anatabine comprises extracting anatabine from the reaction product of Step 2iii using MTBE in a distillation process. The anatabine containing solution is basified to saturation with KOH and K2CO3. Addition of MTBE to the solution induces phase separation, with the anatabine separated into the organic phase, which provides an additional increase in anatabine purity. Subsequent distillation, followed by acid-base work up typically results in an anatabine purity of 99%.

[78] In another aspect, a method of recovering and purifying anatabine comprises extracting anatabine from the reaction product of Step 2iii using MTBE in a distillation process. The anatabine containing solution is basified to saturation with KOH and K2CO3. Addition of MTBE to the solution induces phase separation, with the anatabine separated into the organic phase, which provides an additional increase in anatabine purity. Subsequent distillation, followed by acid-base work up typically results in an anatabine purity of 99%.

[79] Advantageously, this purification approach can be scaled up to industrial-scale manufacture. Purification by chromatography, which does not scale up from smallscale production, is avoided. Moreover, MTBE is more environmentally friendly than halogenated compounds such as chloroform. MTBE also improves extraction efficiency relative to halogenated compounds such as chloroform.

[80] Anatabine prepared according to the methods disclosed herein displays good stability at a variety of temperatures. See, for example, Tables 54 and 55 in Examples 11 and 12, respectively. Purity of the anatabine may be retained for extended periods of time.

2017201593 08 Mar 2017

For example, the anatabine may exhibit the indicated purity at 12 days, at 18 days, at 21 days, at 1 month, at 3 months, at 6 months, at 9 months, at 12 months at 18 months, at 24 months, and at 36 months, or longer. Further, anatabine remains stable when stored at a variety of temperatures. For example, the anatabine retains the indicated purity when stored at -20 °C to -10 °C, -20 °C to 0 °C, -10 °C to 0 °C, 0 °C to 4 °C, 2 °C to 8 °C, 5 °C to 10 °C, 10 °C to 15 °C, 15 °C to 25 °C, or 25 °C to 28 °C. Stability is determined by any suitable art-recognized method. For example, anatabine stability may be monitored by high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LC-MS), or nuclear magnetic resonance (NMR).

[81] The synthesis described herein may yield significantly lower concentrations of byproducts, such as benzophenone, which can lower reaction yields and introduce impurities into the product. Concentrations of benzophenone, for example, are typically less than about 3 % as compared to 8-12 % that were found when reproducing the Deo et al. synthesis. Moreover, yield was 25% for the present invention but only 10% for the Deo et al synthesis.

[82] In an alternative embodiment, methods are provided for synthesizing other minor alkaloids, such as anabasine, nomicotine, N-methylanabasine, or anabaseine. A compound of Formula I may be prepared as previously described and reacted with a dielectrophile to yield a compound of Formula Ila:

[83] The dielectrophile may be selected according to the desired alkaloid. For example, as described in Deo et al., anabasine may be synthesized using 1,4-diiodo-butane; nornicotine may be synthesized using 1,3-diiodo-propane or methanesulfonic acid 4- ethoxy-butyl ester, and so on. The compound of Formula Ila is then acidified as previously described to yield a compound of Formula Illa.

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Formula I

Step 2-i

-----►

Formula Ila

[84] Basification of the compound of Formula Illa results in the desired alkaloid by intramolecular N-alkylation as previously described. The synthetic alkaloids may be provided in a variety of compositions. For example, the compositions may be in the form of a beverage, a chew, a tablet, a lozenge, a gum, and the like. Additional inactive ingredients may be added to improve taste or stability. Optionally, other components such as sweetening and flavoring agents may be added. Other active ingredients may be provided as well.

[85] In some embodiments, an isolated form of anatabine can be provided as one or more pharmaceutically acceptable (or food grade) salts of anatabine. Anatabine may be adsorbed on a cation exchange resin such as polymethacrilic acid (Amberlite IRP64 or Purolite Cl 15HMR), as described in U.S. Patent 3,901,248, the disclosure of which is hereby incorporated by reference in its entirety. Such cation exchange resins have been used commercially in nicotine replacement therapy, e.g., nicotine polacrilex. Unless otherwise clear from context, the term “anatabine” as used herein refers collectively to anatabine and its pharmaceutically acceptable salts. In general, salts may provide improved chemical purity, stability, solubility, and/or bioavailability relative to anatabine in its native form. Non-limiting examples of possible anatabine salts are described in P. H. Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zurich: Wiley-VCH/VHCA, 2002, including salts of l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor- 10-sulfonic acid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid,

2017201593 08 Mar 2017 ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (- L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, pyroglutamic acid (- L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+ L), thiocyanic acid, toluenesulfonic acid (p), and undecylenic acid.

[86] As an alternative to preparing anatabine synthetically, anatabine can be obtained by extraction from tobacco or other plants, such as members of the Solanaceae family, such as datura, mandrake, belladonna, capsicum, potato, nicotiana, eggplant, and petunia. For example, a tobacco extract may be prepared from cured tobacco stems, lamina, or both. In the extraction process, cured tobacco material is extracted with a solvent, typically water, ethanol, steam, or carbon dioxide. The resulting solution contains the soluble components of the tobacco, including anatabine. Anatabine may be purified from the other components of the tobacco using suitable techniques such as liquid chromatography.

[87] As part of the purification process, tobacco material may be substantially denicotinized to remove a majority of other alkaloids such as nicotine, nornicotine, and anabasine. Denicotinizing is usually carried out prior to extraction of anatabine. Methods that may be used for denicotinizing tobacco materials are described, for example, in U.S. Patent 5,119,835, the disclosure of which is hereby incorporated by reference. In general, tobacco alkaloids may be extracted from tobacco material with carbon dioxide under supercritical conditions. The tobacco alkaloids may then be separated from the carbon dioxide by dissolving an organic acid or a salt thereof, such as potassium monocitrate, in the carbon dioxide.

[88] Flue (bright) varieties of tobacco are often used, i.e., Virginia flue. Other tobacco varieties may be used, such as Burley, dark-fired, and/or other commercial tobacco varieties. Methods for forming aqueous tobacco extracts also are known in the art as described, for example, in U.S. Patent 5,065,775, the disclosure of which is hereby incorporated by reference in its entirety. Additional details on methodology for extracting anatabine from tobacco are disclosed in co-pending and commonly owned

U.S. Published Application No. 2010/0154810 Al, the disclosure of which is hereby incorporated by reference in its entirety.

2017201593 08 Mar 2017 [89] In some aspects, a pharmaceutical composition for use in the disclosed methods comprises an isolated form of anatabine and a pharmaceutically acceptable vehicle, diluent, or carrier. An “isolated form of anatabine,” as used herein, refers to anatabine that either has been prepared synthetically or has been substantially separated from plant materials in which it occurs naturally. The isolated form of anatabine as described herein should have a very high purity (including enantiomeric purity in the case of an isolated enantiomer). In the case of synthetic anatabine, for example, purity refers to the ratio of the weight of anatabine to the weight of the end reaction product. In the case of isolating anatabine from plant material, for example, purity refers to the ratio of the weight of anatabine to the total weight of the anatabine-containing extract. Usually, the level of purity is at least about 95%, more usually at least about 96%, about 97%, about 98%, or higher. For example, the level of purity may be about 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or higher. In some aspects, the composition contains levels of other alkaloids, such as nicotine, nomicotine, and anabasine, which are individually about 1 pg/g or less, and which may be about 0.5 pg/g or less, about 0.3 pg/g or less, about 0.1 pg/g or less, or lower.

[90] Anatabine exists in tobacco and other plants as a racemic mixture of R-(+)-anatabine and S-(-)-anatabine, whose structures are illustrated below.

S-(-)-anatabine

2017201593 08 Mar 2017 [91] Methods for selectively preparing the anatabine enantiomers are described, for example, in “A General Procedure for the Enantioselective Synthesis of the Minor Tobacco Alkaloids Nomicotine, Anabasine, and Anatabine,” The AAPS Journal 2005; 7(3) Article 75. Any of the methods or compositions described herein may involve providing anatabine as a racemic mixture of its two enantiomers, as a purified . form of S-(-)-anatabine, or as a purified form of R-(+)-anatabine. Unless otherwise clear from context, the term “anatabine” is used herein to refer to any of (1) a racemic mixture of anatabine (R,S) or a salt thereof, (2) a purified form of S-(-)-anatabine or a salt thereof, or (3) a purified form of R-(+)-anatabine or a salt thereof.

[92] Isolated forms of anatabine or pharmaceutically acceptable salts of anatabine can be provided together with other ingredients, for example, in the form of an elixir, a beverage, a chew, a tablet, a lozenge, a gum, and the like. In one embodiment, for example, a beverage may be in the form of a bottled water product containing about 100 ml to about 2,000 ml purified water and from about 0.00001 to about 0.0001 wt% of a water-soluble salt of anatabine. Additional inactive ingredients may be added to improve product characteristics, such as taste, color/clarity, and/or stability. The bottled water product may also contain other beneficial components, such as vitamins, proteinaceous ingredients, or the like. A composition alternatively may be provided in a solid (e.g., powder) form, such as in a packet, which can be combined with water or other liquid (e.g., by an end user) to prepare a beverage.

[93] Pharmaceutical compositions may be formulated together with one or more acceptable pharmaceutical or food grade carriers or excipients. As used herein, the term “acceptable pharmaceutical or food grade carrier or excipient” means a nontoxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For example, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's

2017201593 08 Mar 2017 solution; ethyl alcohol, and phosphate buffer solutions, as well as compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[94] The pharmaceutical composition may be prepared by any suitable technique and is not limited by any particular method for its production. For example, anatabine can be combined with excipients and a binder, and then granulated. The granulation can be dry-blended any remaining ingredients, and compressed into a solid form such as a tablet.

[95] The pharmaceutical compositions may be administered by any suitable route. For example, the compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or ingested as a dietary supplement or food. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, and intracranial injection or infusion techniques. Most often, the pharmaceutical compositions are readily administered orally and ingested.

[96] The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with acceptable pharmaceutical or food grade acids, bases or buffers to enhance the stability of the formulated composition or its delivery form.

[97] Liquid dosage forms for oral administration include acceptable pharmaceutical or food grade emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylsulfoxide (DMSO) dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

2017201593 08 Mar 2017

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[98J Solid dosage forms for oral administration include capsules, tablets, lozenges, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, acceptable pharmaceutical or food grade excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants such as glycerol, d) disintegrating agents such as agaragar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof, and j) sweetening, flavoring, perfuming agents, and mixtures thereof. In the case of capsules, lozenges, tablets and pills, the dosage form may also comprise buffering agents.

[99] The solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract or, optionally, in a delayed or extended manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Tablet formulations for extended release are also described in U.S. Pat. No. 5,942,244.

Inflammatory Markers [100] Anatabine can be used to reduce elevated blood levels of inflammatory markers such as CRP. Thus, in some embodiments levels of inflammatory markers can be used to aid in determining doses of anatabine to be administered as well as to monitor treatment of various inflammatory disorders and to assist physicians in deciding on a

2017201593 08 Mar 2017 course of a treatment for an individual at risk of an inflammatory disorder. These markers include, but are not limited to, C-reactive protein (CRP), soluble intercellular adhesion molecule (sICAM-1), ICAM 3, BL-CAM, LFA-2, VCAM-1, NCAM, PECAM, fibrinogen, serum amyloid A (SAA), TNFa, lipoprotein associated phospholipase A2 (LpPlA2), sCD40 ligand, myeloperoxidase, interleukin-6 (IL-6), and interleukin-8 (fL-8).

[101] The level of one or more inflammatory markers can be determined in a patient already receiving anatabine therapy or in an individual at risk for an inflammatory disorder or suspected of having an inflammatory disorder. The level is compared to a predetermined value, and the difference indicates whether the patient will benefit from anatabine administration or from continued anatabine administration. The level of inflammatory marker can be determined by any art recognized method. Typically, the level is determined by measuring the level of the marker in a body fluid, for example, blood, lymph, saliva, or urine. The level can be determined by ELISA, or immunoassays or other conventional techniques for determining the presence of the marker. Conventional methods include sending a sample(s) of a patient’s body fluid to a commercial laboratory for measurement.

[102] The predetermined value can take a variety of forms and will vary according to the inflammatory marker. The predetermined value can be single cut-off value, such as a median or a mean, or it can be a range. The predetermined value also can depend on the individual or particular inflammatory disorder. Appropriate ranges and categories can be selected by those of ordinary skill in the art using routine methods. See US 2006/0115903; US 2004/0175754.

[103] Markers such as CRP, sICAM-1, ICAM 3, BL-CAM, LFA-2, VCAM-1, NCAM, PECAM, fibrinogen, SAA, TNF.a, lipoprotein associated phospholipase A2 (LpPlA2), sCD40 ligand, myeloperoxidase, IL-6, and IL-8 are useful markers for systemic inflammation. In some embodiments the inflammatory marker is CRP, which is associated both with cardiovascular disease (see US 2006/0115903) and cancer, such as colon cancer (Baron et al., N. Engl. J. Med. 348, 891-99, 2003). Elevated levels of CRP are also observed in patients with insulin-resistance (Visser et al., JAMA. 1999, 282(22):2131-5). Diabetic and insulin-resistant patients also have elevated levels of

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TNFa, IL-6, and IL-8 (Roytblat et al., Obes Res. 2000, 8(9):673-5; Straczkowski et al., J Clin Endocrinol Metab. 2002, 87(10):4602-6; Hotamisligil et al., Science. 1996,

271(5249):665-8; Sartipy P, Loskutoff D J. Proc Natl Acad Sci USA. 2003,

100(12):7265-70; Hotamisligil et al., J Clin Invest. 1995, 95(5):2409-15).

[104] Each reference cited in this disclosure is incorporated herein in its entirety. The following examples illustrate but do not limit the scope of the disclosure set forth above.

EXAMPLE 1 . NFicB-mediated transcription assays; Cytotoxicity assays [105] The effect of a range of doses of anatabine, nicotine, crude extract of smokeless tobacco, and alkaloid extract of smokeless tobacco was examined in an NFkB luciferase assay (inhibition of TNFa-induced NFkB activity). The smokeless tobacco used in these experiments was plain long-leaf Copenhagen tobacco purchased from a local vendor. Crude extract was extracted with methanol and water and clarified by centrifugation and filtration. The alkaloid extract was prepared from sodium hydroxide and methanol extraction, organic phase separation and purification. AU treatment samples were prepared as a function of weight (pg/ml), and all samples were diluted in DMSO. Dilutions were made immediately before cell culture treatments and, in all cases, the final amount of DMSO did not exceed 1% in cell culture media.

[106] Human endothelial kidney cells (HEK293) transfected with an NFkB luciferase reporter were challenged with TNFa for three hours, then samples were applied to the challenged cells. The results are shown in FIGS. 1-3.

[107] Cytotoxicity assays using the supernatants from the treated cells were conducted using an LDH Cytotoxicity Detection Kit (Roche) according to the manufacturer’s instructions. The results are shown in FIGS. 4-6.

[108] As shown in FIG. 1, TNFa induces an increase in NFicB-mediated transcription of luciferase; administration of anatabine can reduce this transcription to control levels without cellular toxicity (FIG. 4). Crude extracts of smokeless tobacco, while not

2017201593 08 Mar 2017 toxic to cells (FIG. 5), do not reduce TNFa-induced NFicB-mediated transcription (FIG. 2). Although not suitable for administration as pharmaceuticals, both nicotine and an alkaloid extract of smokeless tobacco reduce TNFa-induced NFKB-mediated transcription (FIG. 3); at higher doses, the alkaloid extract demonstrates pronounced cytotoxicity (FIG. 6).

EXAMPLE 2

Materials and methods [109] Animals. Male and female Sprague-Dawley rats (~ 200-250 grams) were obtained from Charles River Laboratories Inc., Wilmington, MA and used in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare. Upon receipt at the vivarium, rats were examined by trained personnel to ensure acceptable health status. Rats were acclimated for at least 5 days prior to use.

[110] Rats were housed 3 per cage. Cage size met or exceeded the requirements set forth by the Institute for Animal Laboratory Research Guide for the Care and Use of Laboratory Animals. The rats were kept in a room maintained at 64-84 °F (22-24 °C) with humidity set at 40-70 %. The room was illuminated with fluorescent lights timed to give a 12 hour-light, 12 hour-dark cycle. Standard rodent diet (PharmaServ lab diet 5001) and water were available for all rats. The feed was analyzed by the supplier, detailing nutritional information and levels of specified contaminants.

[111] Test compounds. The following compounds were tested in the examples below:

(-) Nicotine hydrogen tartrate (Sigma Aldrich: N5260 Lot# 098K0676) 35.1% (w/w) nicotine;

(R, S) Anatabine tartrate (2:3) (Toronto Research Chemicals, A637510, Lot# 9-BHW-79-2) 41.6% (w/w) Anatabine;

(+/-)-nicotine-3'-d3 (Toronto Research Chemicals: N412423, Lot# 9-BCC114-2);

(R,S)-Antabine-2,4,5,6-d4 (Toronto Research Chemicals: A637505, Lot# 6SG-82-l);and

2017201593 08 Mar 2017 anatabine polacrilex (Emerson Resource Inc. lot # JK02-145); purity was 5.18.

% as per Certificate of Analysis |H2] Certificates of analyses for anatabine and nicotine indicated 98% and 100% purity, respectively. Anatabine was stored at 4 °C in a desiccated environment (silica), protected from light. Nicotine was stored at room temperature. Vehicle was sterile phosphate buffered saline (PBS) (Amresco lot#2819B188).

[113] Supplies. The following were obtained from Becton Dickinson, Franklin Lakes, NJ: MICROTAINER® Brand Tubes (K₂) EDTA (lot# 9050883); serum separator blood collection tubes (lot# 9104015); and sodium citrate blood collection tubes (lot# 8310564). Ten percent neutral-buffered formalin was from Sigma Aldrich, St. Louis, MO (batch# 019K4386).

EXAMPLE 3

Toxicokinetic evaluation of single doses of anatabine and nicotine in SpragueDawley Rats [Π4] This example reports evaluation of the toxicokinetics of anatabine and nicotine following a single intravenous injection in Sprague-Dawley rats.

Summary [115] Anatabine was administered as a single intravenous (i.v.) injection at doses of 0.10, 0.75, or 1.0 mg/kg. Nicotine was administered as a single intravenous injection at a dose of 0.4 mg/kg. Six rats (3 males and 3 females) were dosed per dose group. Blood was collected for plasma at 15, 30, 60, 90, 120, 240, 360, 480, and 1440 minutes post i.v. administration. At the 1440 minute time point, animals were euthanized and perfused, and brains were removed and then homogenized. Plasma and brain homogenates were stored at -80 °C until analysis.

[116] An additional 48 rats (24 males and 24 females) received a single intravenous dose via the tail vein at the same doses as mentioned above. At 30 and 360 minutes post administration, 6 rats (3 males and 3 females) per dose group, per time point were euthanized, bled via cardiac puncture, and perfused, and brains were collected. The brains were homogenized. Blood was spun, and plasma was collected. Plasma and brain homogenate were stored at -80 °C.

2017201593 08 Mar 2017 [117] Both anatabine and nicotine can be measured in rat plasma and brain following a single bolus i.v. dose. The concentration of anatabine in plasma is dose-related. Both compounds are also rapidly cleared from plasma; however, the elimination half-life of anatabine is approximately 2- to 2.5-fold greater than that of nicotine (ti/2, 1.64 to 1.68 hr for anatabine compared to 0.67 hr for nicotine). The apparent volume of distribution (Vq) for anatabine is also significantly greater than that of nicotine.

[Π8] At all doses of anatabine the elimination half-life (ti/2), mean residence time (MRT), and exposures (AUCo->«) tended to be higher for female rats compared to male rats; however, only at the highest dose of anatabine (1.0 mg/kg) was this difference statistically significant. At this dose level, the elimination half-life (ti#) of anatabine in females was 1.84 hr compared to 1.44 hr for males; mean residence time (MRT) was 2.80 hr for females compared to 2.18 hr for males; and exposure (AUCo-w) was 788.9 nghr/mL for females compared to 631.3 ng hr/mL for males.

[119] Anatabine and nicotine rapidly appear in brain tissue following i.v. administration, and the concentration of anatabine is dose-dependent. At each dose level the mean concentration of anatabine appeared to be higher in the brains of female animals compared to males; however, the differences were not statistically significant.

[120] Anatabine tartrate (2:3) or nicotine bitartrate was dissolved to the appropriate concentrations in sterile PBS for the i.v. formulations (Table 2). The dosing solutions for each test compound were prepared on the basis of the relative content of the anatabine or nicotine base so that the final concentrations are reflective of the actual base concentration. Four aliquots of each dose level formulation were collected and stored at -80 °C. The test compound, corresponding dose level, number of animals, and sample collection times for Phase I of the study are shown in Table 3. The test compound, corresponding dose level, number of animals, and sample collection times for Phase II of the study are shown in Table 4. The physical signs of each animal were monitored following administration of the test compound.

[121] The animals were weighed prior to dosing and received a single i.v. dose of either test compound at a volume of 5 mL/kg. Blood was collected via the venus plexus (retro-

2017201593 08 Mar 2017 orbital) into tubes containing (K₂) EDTA. No more than 0.5 mL was collected per

I time point. For the 1440-minute time point of Phase I or the 30- and 360-minute time points of Phase II, the animals were euthanized, bled via cardiac puncture, and perfused. The brain was removed, weighed, homogenized in sterile 0.9% saline at a volume equal to its weight, and stored at -80 °C.

[122] Plasma was separated according to the instructions for MICROTAINER® brand collection tubes (3 minutes, 2000x g). Plasma was decanted into microfuge tubes and stored at -80° C. Remaining test compound was stored at -80 °C.

Analytical Methods [123] The signal was optimized for each compound by electrospray ionization (ESI) positive or negative ionization mode. A single ion mode (SIM) scan was used to optimize the Fragmentor for the precursor ion and a product ion analysis was used to identify the best fragment for analysis and to optimize the collision energy. The fragment which gave the most sensitive and specific signal was chosen.

[124] Sample preparation. Plasma and brain samples were treated with three volumes of methanol containing internal standard at 1 μΜ (either (+/-)-nicotine-3'-d3 for nicotine or (7?,5)-Antabine-2,4,5,6-d4 for anatabine), incubated 10 min at 4 °C, and centrifuged. The amount of the test agent in the supernatant was determined by liquid chromatography tandem mass spectrometry (LC/MS/MS).

[125] Analysis. Samples were analyzed by LC/MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 high pressure liquid chromatography (HPLC) and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separation on a hydrophilic interaction liquid chromatography (HILIC) HPLC column (Sepax) using an acetonitrile-ammonium acetate/acetic acid gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in multiple reaction monitoring (MRM) mode. MassHunter software was used to calculate the concentration of the test compounds in samples from the peak area using the appropriate calibration curves.

2017201593 08 Mar 2017 [126] Recovery. Recovery standards were prepared by spiking blank matrix (plasma or brain homogenate) prior to deproteination or after with 23, 62, or 1667 ng/mL of test compound. Deproteination was done by adding 3 columns of methanol containing internal standard with centrifugation to pellet the precipitated protein. Recovery was calculated by dividing the area ratio (peak area of compound over internal standard of the precipitated sample over the recovery standard multiplied by 100. For example: area ratio of spiked plasma/area ratio of spiked deproteinated plasma x 100.

[127] Calibration samples. Calibration curves were determined for both rat plasma and brain homogenate. Calibration samples were prepared by diluting a 50x stock solution

X of the test compound in PBS with blank matrix to the appropriate concentration and these samples were prepared as described above in the sample preparation. Stock solutions were prepared by serial dilution as shown in Table 5.

Results [128] Physical Signs. All males and two females that received nicotine at 0.4 mg/kg experienced tremors immediately post dose and recovered within 2 to 4 minutes. One male (7C) and two females (8A and 8C) in this group also experienced labored breathing which lasted 2 to 4 minutes post dose. The same male (7C) was lethargic and recovered approximately 8 minutes post dose. All other animals in each dose group appeared normal following the administration of the test compounds.

[129] Method Development. Table 6 shows the results of the LC/MS/MS method development for the determination of the appropriate ionization conditions and the mass to charge ratios (m/z) of the parent and product ions for anatabine and nicotine, and their deuterated analogues. The indicated product m/z ratios were used for the analysis of the relevant test samples.

[130] The product ion spectra and sample chromatograms for each compound in Table 6 are shown in FIGS. 12-19. The limits of detection (LOD) of anatabine and nicotine and ' their lower (LLQ) and upper (ULQ) limits of quantitation were derived from the appropriate calibration curves for each test compound and are shown in Table 7.

2017201593 08 Mar 2017 [131] Table 8 provides data on the per cent recovery of each test compound from either rat plasma or brain as a function of the given concentration. Except for the anatabine sample at the LOD and the nicotine samples in rat brain, recovery was generally greater than 90 per cent.

Analysis of Dosing Solutions [132] Table 9 summarizes the analyses of the dosing solutions used in this study. The per cent differences between the actual and expected concentrations are shown. Except for the lowest dose of anatabine, which was 70 % of the expected concentration, the actual concentrations of test compounds were within 20 % of the expected levels.

Plasma Pharmacokinetic Results & Analysis [133] Table 14 lists the plasma concentrations of anatabine and nicotine for all animals at each time point. Table 15 summarizes this data in terms of the mean plasma concentrations of the test compound at each time point for males, females and both genders combined. This data is presented graphically in FIG. 7 and FIG. 8 (semi-log plot). The 24-hr data points from all treatment groups were below the limits of quantitation. Between approximately 6 and 8 hours the plasma concentrations of nicotine and anatabine (0.1 mg/kg) were below the limits of quantitation.

[134] Table 10 and Table 11 provide comparisons for several pharmacokinetic parameters between the different treatment groups and between male and female animals. Both nicotine and anatabine can be measured in rat plasma following a single i.v. bolus, and their concentrations appear to be dose-related. The elimination half-life (ti/2) for each of the anatabine treatment groups was significantly greater than that for the nicotine treatment group (2.1x to 2.5x greater; 0.67 hr for nicotine compared to 1.44 to 1.68 hr for anatabine). The elimination half-lives were similar among the anatabine treatment groups. The longer half-life for anatabine is reflected in the longer mean residence times (MRT), which are about 2-fold longer for anatabine compared to nicotine. Finally, the apparent volume of distribution (Vo) was lower for the nicotine group compared to the anatabine treatment groups. Amongst the anatabine treatment groups, Vd was significantly greater for the 0.1 mg/kg dose group compared to either

2017201593 08 Mar 2017 of the two higher doses; however, it is not known whether this is a real difference or whether it is due to variability and the fewer number of measurable data points at the low dose.

[135] Table 11 shows a comparison of these same parameters between male and female rats within each treatment group. There were no statistically significant differences between males and females except in the highest anatabine treatment group (1.0 mg/kg) where the females exhibited a longer elimination half-life and therefore, longer mean residence time than the males (ti/2, 1.84 ± 0.16 hr and MRT, 2.80 ± 0.24 hr, females compared to ti/2, 1.44 ± 0.08 and MRT, 2.18 ± 0.12 hr, males). This difference is apparent for all treatment groups, although it only achieved statistical significance in the highest anatabine group. The females in this treatment group also displayed a much greater overall exposure (AUCo-.») to anatabine than the male animals. This difference is depicted in FIG. 9, which shows the dose-exposure relationship for anatabine and nicotine. Overall, there appears to be a linear response between dose and exposure for anatabine; it is not possible to determine if the female animals display a non-linear response at high doses of anatabine.

[136] FIG. 11 shows the dose-concentration response for the 0.5-hour time point for males and females at each dose level. It appears that the brain levels of anatabine begin to level off between 0.75 mg/kg and 1.0 mg/kg.

[137] Table 14 lists the concentrations of anatabine and nicotine in the brain extracts for all animals at each time point. Table 15 summarizes this data in terms of the mean concentrations of the test compound per gram of brain tissue at each time point for males, females and both genders combined. This data is presented graphically in FIG. 10 and in tabular form in Table 12. After the 6-hour time point most concentrations were below the limits of quantitation; however, the test compound concentration was quantifiable in several samples at 24-hours.

[138] FIG. 11 shows the dose-concentration response for the 0.5-hour time point for males and females at each dose level. It appears that the brain levels of anatabine begin to level off between 0.75 mg/kg and 1.0 mg/kg.

Discussion

2017201593 08 Mar 2017 [139] ' All males and two females that received nicotine at 0.4 mg/kg experienced tremors immediately post dose; however they recovered within 2 to 4 minutes. One male (7C) and two females (8A and 8C) in this group also experienced labored breathing which lasted 2 to 4 minutes post dose. The same male (7C) was lethargic and recovered approximately 8 minutes post dose. All animals in each of the anatabine dose groups appeared normal immediately following administration of the test compounds and no obvious adverse signs were observed.

[140] Both nicotine and anatabine can be measured in rat plasma following a single, bolus, i.v. dose and their concentrations appear to be dose-related. The elimination half-life of anatabine is approximately 2- to 2.5-fold greater than that of nicotine, and this is also reflected in a longer mean residence time, which is approximately twice as long as that for nicotine. The 24-hr data points from all treatment groups were below the limits of quantitation and it appears that at the doses selected, the test compounds are cleared from rat plasma between 8 and 24 hours post-administration.

[141] The apparent volume of distribution (Vq) was also significantly lower for the nicotine group compared to the anatabine treatment groups. Amongst the anatabine treatment groups, Vd was significantly greater for the 0.1 mg/kg dose group compared to either of the two higher doses; however, it is not known whether this is a real difference or whether it is due to variability and the fewer number of measurable data points at the low dose.

[142] When comparisons between male and female animals were conducted for these same parameters, within each treatment group, there were no statistically significant differences observed except for the highest anatabine treatment group (1.0 mg/kg) where the females exhibited a longer elimination half-life and therefore, longer mean residence time than the males (tj/2, 1.84 ± 0.16 hr and MRT, 2.80 ± 0.24 hr, females compared to ti/2, 1.44 ± 0.08 and MRT, 2.18 ± 0.12 hr, males). In fact, these differences between male and female animals were apparent for all treatment groups, although statistical significance was achieved only at the highest anatabine dose tested. The females in this treatment group also displayed a much greater overall exposure (AUCo->_M) to anatabine than the male animals. Overall, there is a linear response between dose and plasma concentrations or exposure to anatabine in both male and female rats; although the response appears to be somewhat greater in female

2017201593 08 Mar 2017 animals and is more pronounced at the higher dose levels. It is not possible to determine from the data if the female animals display a non-linear response at higher doses of anatabine.

[143] Both anatabine and nicotine rapidly appear in brain tissue following i.v. administration. The concentrations of anatabine are dose-dependent but appear to level off between 0.75 mg/kg and 1.0 mg/kg. This observation is based on the levels measured only at the 0.5-hour time point and a greater number of time points are required for a more thorough evaluation. There were no statistically significant differences in the concentrations of either test compound in brain between male and female animals; however at each dose level the mean concentrations in the brains of females tended to be somewhat higher.

EXAMPLE 4

Toxicokinetic evaluation of single doses of anatabine and nicotine with a 14-day observation period [144] This example reports the evaluation of the toxicity of anatabine or nicotine for a period of fourteen days following a single intravenous injection in Sprague-Dawley rats. The toxicity of anatabine and nicotine was evaluated after a single intravenous (i.v.) injection in the rat. Anatabine was administered as a single intravenous injection at doses of 0.10, 0.75, or 1.5 mg/kg. Nicotine was administered as a single intravenous injection at a dose of 1.50 mg/kg. One control group of animals received a single i.v. dose of the vehicle at 5 mL/kg. Ten rats (5 males and 5 females) were dosed per group. Due to animal mortality in the nicotine-dosed group, the surviving animals were taken off study and a separate nicotine tolerability study was conducted. One female received a single i.v. dose of 1.25 mg/kg, and 3 females received a single i.v. dose of 1.0 mg/kg. Following the tolerability study, a group of 5 males and 5 females received a single i.v. dose of nicotine at 0.75 mg/kg.

[145] All rats dosed with vehicle or anatabine, and the animals dosed with 0.75 mg/kg of nicotine were observed daily for 14 days. Body weight and food consumption was measured daily for 14 days. On day 15, urine was collected on all surviving animals. The animals were euthanized and bled via cardiac puncture, and blood was collected

2017201593 08 Mar 2017 for analysis. Tissues were collected, weighed, evaluated for gross abnormalities, and stored in 10% neutral-buffered formalin.

[146] All groups appeared normal immediately after dosing except for the animals dosed with 1.5 mg/kg of anatabine and those dosed with 1.5 mg/kg of nicotine. Both males and females dosed with 1.5 mg/kg of anatabine experienced tremors upon compound administration. The animals appeared normal by 15 minutes post dose. Upon completion of the 1.5 mg/kg dose of nicotine, tremors and rigidity were observed in all dosed animals. The tremors were more severe in the females. One male did not survive, whereas the other 4 appeared normal after 15 minutes. Three females were dosed and two died within 5 minutes of dosing; the remaining 2 females were not dosed due to the morbidity in the group. The surviving animals from this group were removed from study. These results suggest that both anatabine and nicotine affect both the peripheral and central nervous systems.

[147] During the tolerability study, all rats (1 female dosed with 1.25 mg/kg of nicotine and 3 females dosed with 1.0 mg/kg of nicotine) experienced severe tremors upon completion of dosing, but all returned to normal by 20 minutes post dose. These animals were not included in the 14-day observation period.

[148] Both males and females dosed with nicotine at 0.75 mg/kg experienced tremors upon compound administration but returned to normal within 15-20 minutes post dose. One male and two females died post dose. Surviving animals in all groups appeared normal throughout the 14-day observation period. The body weights for both male and female rats in the nicotine group were lower than those in the control and anatabine treatment groups; however, these were still within the study-specified range. Consequently body weight gain for this treatment group was also somewhat lower than the vehicle controls. Food consumption was similar among the groups over the 14-day period; however, consumption by males treated with 0.1 mg/kg or 1.5 mg/kg anatabine appeared to be somewhat higher than animals in the control group. This is not considered to be a treatment-related effect.

[149] Hematology and blood chemistries for male and female animals were analyzed and evaluated for differences between the individual treatment groups and the relevant vehicle controls. All treatment groups showed no significant differences relative to the

2017201593 08 Mar 2017 controls and/or the values were well within the normal ranges expected for this species. Similarly, no notable differences in any of the urinalysis parameters were observed between animals treated with either anatabine or nicotine, relative to the controls.

[150] Anatabine or nicotine was dissolved to the appropriate concentrations in sterile PBS for the i.v. formulations (see Table 16). The dosing solutions for each test compound were prepared on the basis of the relative content of the anatabine or nicotine base so that the final concentrations reflect the actual base concentrations. Four aliquots of each dose formulation were collected and stored at -80 °C. The test compound, corresponding dose level, number of animals, and frequency of observations are shown in Table 17.

[151] The animals were weighed prior to dosing and received a single i.v. dose via the lateral tail vein of either test compound or vehicle at a volume of 5 mL/kg. Due to animal mortality in the nicotine-dosed group (1.5 mg/kg), the surviving animals were taken off study and a separate nicotine tolerability study was conducted.

Nicotine Tolerability Study

11521 One female rat was dosed intravenously with 1.25 mg/kg of nicotine, and three females were received 1.0 mg/kg intravenously. Following the tolerability study, an additional group was added to the study. Five males and five females received a single intravenous dose of nicotine at 0.75 mg/kg. All animals were observed daily. Body weight and food consumption was measured daily, with any abnormal observations noted. Average daily body weights and food consumption was tabulated with standard deviation calculated.

[153] On day 15, urine was collected on all surviving animals for urinalysis. The animals were euthanized and bled via cardiac puncture. Blood was collected for hematology, clinical chemistry, and coagulation analysis. Tissues were collected, weighed, and stored in 10% neutral-buffered formalin for possible future analysis. The tests and tissues collected are summarized in Table 18.

2017201593 08 Mar 2017

Results

Dosing Solution Analysis [154] Table 19 summarizes the dosing solutions used during the conduct of this study. The per cent differences between the actual and expected concentrations of the test compounds are shown. The actual concentrations were within 20 per cent of the expected levels.

General Observations [155] All groups appeared normal immediately after dosing except for the animals dosed with 1.5 mg/kg of anatabine and those dosed with 1.5 mg/kg of nicotine. Both males and females dosed with 1.5 mg/kg of anatabine experienced tremors upon compound administration. The animals appeared normal by 15 minutes post dose. Following administration of the 1.5 mg/kg dose of nicotine, tremors and rigidity were observed in all animals. The tremors were more severe in the females. One male did not survive, whereas the other 4 appeared normal after 15 minutes. Three females in this group were dosed and two died within 5 minutes of dosing; the remaining 2 females were not treated due to the observed morbidity in the group. The surviving animals from this group were removed from the study.

[156] During the tolerability study, all rats (1 female dosed with 1.25 mg/kg of nicotine and 3 females dosed with 1.0 mg/kg of nicotine) experienced severe tremors upon completion of dosing, but all returned to normal by 20 minutes post dose. These animals were not included in the 14-day observation period.

[157] Both males and females dosed with nicotine at 0.75 mg/kg experienced tremors upon compound administration, but returned to normal within 15-20 minutes post dose. One male and two females died post dose.

[158] Surviving animals in all groups appeared normal throughout the 14-day observation period.

Body Weights, Growth Rates and Food Consumption

2017201593 08 Mar 2017 [159] The daily measured body weights for each animal are tabulated in Tables 28A-F and the average daily food consumption is summarized in Tables 29A, B. These data are summarized in Table 20 for the average weight gain over the 14-day observation period and the average daily food consumption, by treatment group and gender. FIG. 20 shows the mean body weights of animals in each treatment group on the day of dosing (Day 0) and for each day, thereafter.

[160] The average weight gains for animals in each treatment group over the 14-day observation period were similar to those in the vehicle control group, except for the nicotine-dosed group of male animals that exhibited weight gains that were significantly lower than the controls. The mean increase in the weight of females of the nicotine-dosed group was also lower than that of the vehicle control, though not statistically significant at the 5 per cent level. It should be noted that the mean weights of the male and female animals in the nicotine-treated group at Day 0 were lower than their corresponding genders in the vehicle control. The difference for males was statistically significant (Vehicle: 234.6 ± 9.9 g versus Nicotine: 216.0 ± 6.2 g; /7=0.014), although that for females was not (Vehicle: 209.8 ± 7.3 g versus Nicotine: 195.3 ± 10.4 g;/7=0.058).

[161] The average daily food consumption per animal was statistically higher in the males of the 0.1 mg/kg and 1.5 mg/kg anatabine treatment groups. This difference is not considered to be clinically significant or related to any treatment effects.

[162] Overall, although some differences in the changes in weight and food consumption were statistically significant, they are not considered to be treatment-related.

Necropsy Observations and Organ Weights [163] Upon necropsy and organ collection no noticeable differences or abnormalities were observed between the vehicle-dosed animals and the test compound-dosed animals. Individual organ weights can be found in Table 36. Several statistically significant differences in organ weights were noted (see Table 21 and Table 22); however, they do not appear to be dose-related and likely due to the small sample sizes and

2017201593 08 Mar 2017 variability in the organ collection. In general, several organ weights tended to be lower in the nicotine-treated group, although this observation is likely related to the lower animal weights in this group relative to the controls.

Hematology and Coagulation Parameters

11641 Plasma samples collected for hematology were analyzed, and individual values for the various parameters for each animal are listed in Table 31 (normal ranges, Table 30) and these are summarized in terms of descriptive statistics in Table 23A, Table 23B, and Table 24. Also shown are statistical comparisons between the vehicle controls and the various treatment groups, subdivided by gender.

[165] In general, there were few significant differences between the treatment groups and the vehicle control group for either gender. Female rats in 0.1 mg/kg anatabine group showed a small but statistically significant decrease in mean corpuscular hemoglobin concentration (MCHC) relative to the control; however, the values are still within the normal range for this species. Similarly, females in the 1.5 mg/kg anatabine and 0.75 mg/kg nicotine treatment groups showed small, but statistically significant decreases in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), although these values were still within the normal range for this species as well.

1166] Males and females in the 0.75 mg/kg and 1.5 mg/kg anatabine groups showed a statistically significant decrease in reticulocyte count compared to the control animals; however, these values are also well within the normal range for this parameter.

(167] There were no notable differences in red blood cells, white blood cells, platelet counts, lymphocyte, monocyte, eosinophil and basophil counts, or neutrophil segmentation.

[168] Individual values for the coagulation parameters activated partial thromboplastin (aPTT) and prothrombin times (PT) for each animal are listed in Table 34 (normal ranges, Table 30). These are summarized in terms of descriptive statistics in Table 25. Also shown are statistical comparisons between the vehicle controls and the various treatment groups, subdivided by gender. There were no significant differences in aPTT or PT between the vehicle control and each of the anatabine treatment groups;

2017201593 08 Mar 2017 although the aPTT values for all these groups were outside the normal range. In both male and female animals of the nicotine group, however, aPTT was significantly lower relative to the vehicle control group, indicative of faster clotting times due to the intrinsic, contact activation pathway. The origin of this difference is not known, although the values are within the normal range for this species.

Clinical Chemistry [169] Plasma samples collected for blood chemistries were analyzed, and individual values for the various parameters for each animal are listed in Table 33 (normal ranges, Table 32), and these are summarized in terms of descriptive statistics in Tables 26A, 26B, 27A, and 27B. Also shown are statistical comparisons between the vehicle controls and the various treatment groups, subdivided by gender.

[170] Values for all clinical chemistry parameters were within the respective normal ranges. There were several parameters where statistically significant differences were noted between treatment groups and controls. Specifically, males treated with anatabine at 0.75 mg/kg and 1.5 mg/kg showed slight increases in albumin levels, as did females treated with 0.1 mg/kg and 0.75 mg/kg anatabine, but not at 1.5 mg/kg. Total protein was slightly increased in males in all anatabine treatment groups and the nicotine group relative to vehicles controls. In females, total protein was somewhat higher only in the 0.1 mg/kg anatabine and nicotine groups. Finally, as with total protein, globulins were marginally higher at all anatabine dose levels and the nicotine dose group in males. Globulins were also slightly higher in females in the 0.1 mg/kg anatabine and nicotine groups. The higher globulin levels, but not albumin, in the nicotine group is reflected in slightly lower A/G ratios, for both genders. Nevertheless, all the reported values for albumin, globulins and total protein were within the normal range for this species. There were small, but statistically significant differences noted for calcium levels in males in the nicotine-treated group and for sodium levels in males at 0.75 mg/kg and 1.5 mg/kg anatabine and females in the 1.5 mg/kg anatabine treatment groups. The values are well within normal ranges and therefore, not clinically significant.

2017201593 08 Mar 2017

Urinalysis

1171] Individual values of the urinalysis parameters for each animal are listed in Table 35.

There were no notable differences between the active treatment groups and controls and the observations are all consistent with those expected for this species.

Discussion [172] The toxicity of anatabine and nicotine was evaluated after a single intravenous (i.v.) injection in the rat. Anatabine was administered at doses of 0.10, 0.75, or 1.5 mg/kg. Nicotine was administered at a dose of 1.50 mg/kg, initially; however, due to mortality and significant adverse effects observed at this dose and at lower doses of 1.0 mg/kg and 1.25 mg/kg, a separate group was included in the study and dosed with nicotine at 0.75 mg/kg. One group of animals received a single i.v. dose of the vehicle at 5 mL/kg. Ten rats (5 males and 5 females) were dosed per group.

[173] All rats dosed with vehicle or anatabine, and the animals dosed with 0.75 mg/kg of nicotine were observed daily for 14 days. Body weight and food consumption was measured daily for 14 days. On day 15, urine was collected on all surviving animals. The animals were euthanized, bled via cardiac puncture, and blood was collected for analysis. Tissues were collected, weighed, any gross abnormalities were noted, and stored in 10% neutral-buffered formalin for possible future analysis.

[174] All animals, at all dose levels of anatabine, survived the study; however, those in the 1.5 mg/kg anatabine group experienced tremors and shaking immediately after test compound administration, which lasted for approximately 15 minutes post-treatment. In the nicotine treatment group (0.75 mg/kg), one male animal and 2 females died following test compound administration, and all animals experienced tremors and shaking for up to 20 minutes post-administration. These results suggest that both anatabine and nicotine affect both the peripheral and central nervous systems.

[175] The growth rates and food consumption in all anatabine treatment groups were similar to their appropriate male or female vehicle controls. Male rats in the nicotine treatment group had a slightly lower growth rate; however, this is unlikely to be related to the test compound. This group of animals began the study at a lower

2017201593 08 Mar 2017 average weight than males in the control or anatabine treatment groups. The food consumption in males, in the 0.1 mg/kg and 1.5 mg/kg anatabine groups was somewhat higher than controls and although the result was statistically significant it is not likely to be related to an effect of the test compound.

[176] At necropsy, no noticeable differences or gross abnormalities were observed in any of the organs collected between the vehicle-treated and the test compound-treated animals. Several statistically significant differences in organ weights^were noted; however, they do not appear to be dose-related and are likely due to the small sample sizes and the inherent variability associated with organ collection. The weights of heart, liver and kidneys in males, and thymus and heart in females of the nicotinetreated group were significantly lower than those of the corresponding vehicle controls; however, this observation is likely related to the lower overall animal weights in this group relative to the controls.

[177] The hematology parameters for all treatment groups and genders were within the normal ranges expected for this species or displayed no significant differences when compared to the vehicle controls. Activated partial thromboplastin and prothrombin times were similar for all anatabine treatment groups relative to the controls; however, they were higher than the expected normal range. Both males and females in the nicotine group displayed significantly shorter clotting times via the intrinsic or contact activation pathway (aPTT) compared to the relevant control animals; however, the values were within the normal ranges for this species. Clotting times via the extrinsic or tissue factor pathway as determined by prothrombin times (PT) were normal.

[178] Values for all clinical chemistry parameters were within the respective normal ranges or showed no differences relative to the vehicle control group.

[179] Evaluation of the individual urinalysis parameters for each animal showed no notable differences between the active treatment groups and controls.

2017201593 08 Mar 2017

EXAMPLE 5

Toxicokinetic evaluation in Sprague-Dawley rats of oral multi-dose administration of anatabine

1180] This example reports the results of an evaluation of the pharmacokinetics of anatabine following multiple oral doses in Sprague-Dawley rats.

Summary [181] The plasma pharmacokinetic profile of orally administered anatabine was investigated in the rat. This study consisted of two groups of 8 animals each, 4 males and 4 females. One group received a total of 0.6 mg anatabine per kilogram body weight (BW) and the second group received 6.0 mg anatabine per kilogram BW in three, divided, oral, doses of 0.2 mg/kg BW (0.6 mg total) or 2.0 mg/kg BW (6.0 mg total). The test compound was administered as anatabine polacrilex and each dose was administered at 0, 4, and 8 hours and was administered in a volume of 5 mL/kg BW. Blood was collected for plasma at 30, 60, 240, 270, 300, 480, 540, 600, 720 and 1440 minutes post initial dose.

[182] All animals in both treatment groups appeared normal immediately following each administration of the test compound and no adverse signs were observed for the duration of the observation and plasma sampling period.

[183] The mean time to maximal plasma concentration following the first two oral doses ranged from 0.50 to 0.88 ± 0.25 hr. There were no significant differences between gender or dose group. After the third dose of test compound, the mean time to maximal plasma concentration ranged from 1.00 to 2.00 ±1.41 hr. Within each dose group there were no significant differences in C_p, _max between males and females and nor was there any significant change in this parameter over time. In females of the high dose group C_{p> max} appeared to increase from 259.8 ± 35.4 ng/mL to 374.8 ± 122.9 ng/mL; however, the trend was not statistically significant.

[184] There were two, observable, minima following the first two oral doses of anatabine polacrilex. In general, the minima were not significantly different from one another over time, except for females of the high dose group, which increased from 51.5 ± 26.0 ng/mL to 180 + 31 ng/mL.

2017201593 08 Mar 2017

1185] The total exposure, elimination half-lives, mean transit times and mean absorption times did not differ significantly between male and female rats within the two treatment groups. When these data are combined and grouped according to dose level the total exposure is significantly greater at the high dose as would be expected; however, the terminal elimination half-life is also significantly higher in the 6.0 mg/kg BW group compared to the 0.6 mg/kg BW dose group.

1186] The overall elimination half-life of anatabine following the first oral dose was 1.93 ± 0.73 hr, the mean transit time was 3.01 ± 1.25 hr and the mean absorption time was 0.56 ± 1.25 hr. The mean absorption time of 0.56 compares favorably with the calculated T_max values following the first two doses and indicates that the absorption of anatabine occurs within the first 30 to 60 minutes after oral administration.

[187] Anatabine was stored at 4 °C, protected from light. The vehicle was sterile phosphate buffered saline (PBS) (Amresco).The test compound was formulated in sterile phosphate buffered saline (PBS) based on the content of anatabine base in the anatabine polacrilex. Two formulations were prepared; one for each of the two treatment groups. The test compound was formulated for each treatment group just prior to the first dose administration and constantly stirred until dosing was completed (Table 37). Four aliquots of each dose level formulation were collected and stored at 80 °C. The test compound, corresponding dose level, and number of animals are shown in Table 38. The sample collection times are shown in Table 39.

[188] The physical signs of each animal were monitored following administration of the test compound.

[189] The animals were weighed prior to dosing and received three doses p.o. of test compound at a volume of 5 mL/kg. Blood was collected via the venus plexus (retroorbital) into tubes containing (K₂) EDTA. No more than 0.5 mL was collected per time point. For the 1440-minute time point the animals were euthanized, and bled via cardiac puncture.

[190] Plasma was separated as per package instructions for MICROTAINER® brand collection tubes (3 minutes, 2000x g). Plasma was decanted into microfuge tubes and stored at -80° C. Remaining test compound was placed at -80 °C .

2017201593 08 Mar 2017 [191] Sample preparation. Plasma samples were treated with three volumes of methanol containing internal standard at 1 μΜ (J?,5)-Antabine-2,4,5,6-d4), incubated 10 min at °C, and centrifuged. The amount of the test agent in the supernatant was determined by LC/MS/MS.

1192] Analysis. Samples were analyzed by LC/MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 high pressure liquid chromatography (HPLC) and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separation on a Hydrophilic interaction liquid chromatography (HILIC) HPLC column (Sepax) using an acetonitrile-ammonium acetate/acetic acid gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in multiple reaction monitoring (MRM) mode. MassHunter software was used to calculate the concentration of the test compounds in samples from the peak area using the appropriate calibration curves.

[193] Calibration samples. Calibration curves were determined in rat plasma. Calibration samples were prepared by diluting a 50x stock solution of the test compound in PBS with blank matrix to the appropriate concentration and these samples were prepared as described above in the sample preparation. Stock solutions were prepared by serial dilution as shown in Table 40.

[194] Data Analysis. Descriptive statistics were calculated for all pharmacokinetic parameters. Elimination half-lives (ti^) were calculated by linear regression of logarithmically transformed plasma concentration data for each period between doses and following the final dose.

[195] Total areas under the plasma concentration curves (AUC) and under the first moment curves (AUMC) were calculated using linear trapezoidal summation across all concentration time points as well as for intervals between each dose administration and following the final dose. For the interval following the first oral dose of anatabine polacrilex, mean transit times (MTT) were calculated from the corresponding ratio of AUMC to AUC. Mean absorption times (MAT) were calculated according to the following relation:

MAT = MTT - MRT,

2017201593 08 Mar 2017 [196] where MRT represents the mean residence time. This was calculated from the mean residence times.

[197] The statistical comparison of parameters between male and female animals was made using a two-tailed, unpaired, ί-test with a 95 per cent confidence interval. Repeatedmeasures analysis of variance (ANOVA) was used for multiple comparisons of C_Pimax involving successive determinations on the same group of animals.

Results [198] Physical Signs. No adverse events were observed.

[199| Method Development. Table 41 shows the results of the LC/MS/MS method development for the determination of the appropriate ionization conditions and the mass to charge ratios (m/z) of the parent and product ions for anatabine and its deuterated analogue as determined above. The indicated product m/z ratios were used for the analysis of the relevant test samples.

[200] See Example 3 for the product ion spectra and sample chromatograms for each compound in Table 41. The limits of detection (LOD), lower (LLQ), and upper (ULQ) limits of quantitation was derived from the calibration curve and are shown in Table 42.

[201] Analysis of Dosing Solutions. Table 43 provides a summary of the analyses of the dosing solutions used during the conduct of this study. The per cent differences between the actual and expected concentrations are shown. The lowest dose of anatabine, which was 63% of the expected concentration and the high dose was 84% of the expected level.

[202] Plasma Pharmacokinetic Results & Analysis. FIG. 21A and FIG. 21B show the mean plasma anatabine concentration-time curves for male and female rats in each of the two dose groups: 0.6 mg/kg (FIG. 21A) and 6.0 mg/kg BW (FIG. 21B). FIG. 22A and FIG. 22B show the same data with the values from both males and females combined. In each instance, three plasma concentration maxima can be observed corresponding to the administration of the three divided doses of anatabine polacrilex

2017201593 08 Mar 2017 at 0, 4 and 8 hours. Similarly, two anatabine plasma concentration minima are found prior to administration of the final dose.

[203] The mean maxima and minima anatabine plasma concentrations (C_{p> ma}x, C_{p> m}i_n) for males and females in each dose group are recorded in Table 44 along with the mean time to maximal concentration following each of the three doses (T_max ). Statistical comparisons between male and female animals within each dose group revealed no significant differences in any of the parameters, except for the second plasma concentration minimum (C_p,_mi_n(2)) in both treatment groups; 15.3 ± 5.5 ng/mL versus 7.5 ± 1.7 ng/mL in the 0.6 mg/kg BW treatment group, and 93 ± 16 ng/mL versus 180 ±31 ng/mL in the 6.0 mg/kg BW treatment group. FIG. 23A and FIG. 23B show the data in Table 44 plotted as a function of time.

[204] The times to reach maximal concentration generally occurred within 0.5 hr and 1.0 hr post administration in both treatment groups and for both genders, following doses one and two (see Table 45). After the third dose, t_max(3) was generally between 1.0 and 2.0 hours post-administration; however, it should be noted that the earliest sampling point was at 1 hr following this dose.

[205] Table 45 shows a comparison of the plasma concentration maxima and minima over time for male and female rats in both treatment groups. There were no statistically significant changes in any of these parameters except for the plasma concentration minima for female rats in the high dose group; C_{p> m}j_n increased from 51.5 ± 26.0 ng/mL to 180.0 ± 30.7 ng/mL.

[206] The mean exposures (AUC), elimination half-lives (ti/2), mean transit times (MTT) and mean absorption times (MAT) are reported in Table 46 for male and female animals in the two treatment groups. There are no significant differences between the genders in any parameter, at either dose level.

[207] When the male and female data are combined, as shown in Table 47, there is a significant difference in total exposure as would be expected as a consequence of the two different dose levels (AUCo-+«>; 285 + 77 nghr/mL versus 3496 ± 559 nghr/mL). There is also a significant difference in the terminal elimination half-life between the two treatment groups (ti/2, terminal; 1.79 ± 0.64 hr versus 4.53 ± 1.77 hr), where ti/2.

2017201593 08 Mar 2017 terminal refers to the elimination half-life following the final dose of anatabine polacrilex.

[208] As there were no significant differences in the calculated elimination half-life, mean transit times and mean absorption times between treatment groups following the first dose of the test compound (tj/2, o->4, MTTo-»4, and MATo-,4, respectively), the data at both dose levels were combined for males and females (see Table 48). There were no significant differences in these parameters between genders.

[209] Table 49 provides animal weights and dosing times. Table 50 provides measured concentrations of anatabine in rat plasma samples at each time point. Table 51 provides mean concentration and description statistics of anatabine in plasma samples at each time point.

12101 The data from both genders are also combined to give corresponding overall values. The calculated mean elimination half-life (ti/2, o->4) is 1.93 ± 0.73 hr, the mean transit time (MTTo_>4) is 3.01 ± 1.25 hr, and the mean absorption time (MATo-^) is 0.56 ± 1.25 hr.

Discussion [211] This study evaluated the pharmacokinetics of anatabine in male and female SpragueDawley rats following the repeat-dose administration of anatabine polacrilex by oral gavage at two different dose levels. Anatabine was administered at 0.6 mg/kg BW in three, divided, doses of 0.2 mg/kg BW, or at 6.0 mg/kg BW in three, divided, doses of 2.0 mg/kg BW. Each dose was separated by an interval of four hours. All animals in both treatment groups appeared normal immediately following each administration of the test compound and no adverse signs were observed for the duration of the observation and plasma sampling period.

[212] Anatabine concentrations can be measured in rat plasma following single and repeat oral dosing. The mean time to maximal plasma concentration following the first two oral doses ranged from 0.50 to 0.88 ± 0.25 hr. There were no significant differences between gender or dose group. After the third dose of test compound, the mean time to maximal plasma concentration ranged from 1.00 to 2.00 ± 1.41 hr; although in this instance the first time point measured was at one hour post-dose and therefore, it is

2017201593 08 Mar 2017 possible that actual maximum occurred prior to this time. Within each dose group there were no significant differences in C_p, _max between males and females, nor was there any significant change in this parameter over time. In females of the high dose group C_p, _max appeared to increase from 259.8 ± 35.4 ng/mL to 374.8 ± 122.9 ng/mL;

however, the trend was not statistically significant.

[213] There were also two, observable, minima following the first two oral doses of anatabine polacrilex. In general, the minima were not significantly different from one another over time, except for females of the high dose group, which increased from 51.5 ± 26.0 ng/mL to 180 ± 31 ng/mL. Overall, these results suggest that with a 4 hour dosing interval, and after eight hours, near steady-state conditions appear have been achieved in male animals, whereas in females this may not yet be the case.

[214] Within the two treatment groups, the total exposure, elimination half-lives, mean transit times and mean absorption times did not differ significantly between male and female rats. When these data are combined and grouped according to dose level the total exposure is significantly greater at the high dose as would be expected; however, the terminal elimination half-life is also significantly higher in the 6.0 mg/kg BW group compared to the 0.6 mg/kg BW dose group. The reason for this difference is not apparent since the mean transit times and mean absorption times did not differ significantly.

[215] The elimination half-life, mean transit time and mean absorption time following the first oral dose of the test compound are the most reliable estimates of these parameters since the plasma concentration data are not confounded by carry-over amounts from a previous dose. The overall elimination half-life of anatabine following the first oral dose was 1.93 ± 0.73 hr, the mean transit time was 3.01 ± 1.25 hr and the mean absorption time was 0.56 + 1.25 hr. The mean absorption time (also often called mean arrival time) of 0.56 compares favorably with the calculated T_max values following the first two doses and indicates that the absorption of anatabine occurs within the first 30 to 60 minutes after oral administration.

2017201593 08 Mar 2017

EXAMPLE 6

Treatment of thyroiditis [216] This example illustrates administering anatabine for treating thyroiditis. A female patient, aged approximately 52, had been afflicted with Hashimoto’s thyroiditis for approximately 5 years. The patient’s condition had advanced to a state where the treating physician recommended a thyroid lobectomy. The patient orally ingested a tablet containing about 600 pg anatabine citrate, 20 times daily over a period of 30 days. At the conclusion of the treatment, inflammation of the thyroid was reduced to normal levels, such that the patient was no longer in need of a thyroid lobectomy. The patient continued the treatment for an additional 30 days, after which time the patient’s voice distortion associated with thyroiditis was no longer present.

2017201593 08 Mar 2017

Table 1.

headaches	Reuter et al. (2006) Headache 43: 426-427
pain	Tegeder et al. (2004) Journal of Neuroscience 24: 16371645 Niederberger et al. (2004) Neurosci. 24, 1637-45 Niederberger et al. (2008) FASEB Journal 22:3432-3442
complex regional pain syndrome	Hettne et al. (2007) Journal of Biomedical Discovery and Collaboration 2: 2
cardiac hypertrophy	Purcell & Molkentin (2003) Circulation 108: 638-640 Freund et al. (2005) Circulation 111: 2319-2325 Sen & Roy (2005) American Journal of Physiology: Heart and Circulatory Physiology 289: H17-H19
muscular dystrophy (type 2a)	Baghdiguian et al. (1999) Nature Medicine 5: 503-511 ; erratum in Nature Medicine 1999 Jul;5(7):849]
muscle wasting	Hasselgren (2007) Journal of Cellular Physiology 293: R1545-R1551
catabolic disorders	Holmes-McNary (2002) Current Opinion in Clinical Nutrition and Metabolic Care 5: 255-263
diabetes mellitus, type 1	Ho & Bray (1999) Proceedings of the Society of Experimental Biology and Medicine 222: 205-213 Eldor et al. (2006) Proceedings of the National Academy of Sciences USA 103: 5072-5077
diabetes mellitus, type 2	Yuan et al. (2001) Science 293: 1673-1677 Lehrke et al. (2004) PLoS Medicine 1: e45 Chen F (2005) Biochemical and Biophysical Research Communications 332: 1-3
obesity	Gil et al. (2007) British Journal of Nutition 98: S121-S126
fetal growth retardation	Mammon et al. (2005) Review of Diabetic Studies 2 27-34
hypercholesterolemia	Wilson et al. (2000) Atherosclerosis 148: 23-30
atherosclerosis	Brand et al., J Clin Invest 1996;97:1715-1722. Ross et al. (2001) American Journal of Clinical Pathology 116: S97-S107 Li & Gao (2005) Medical Hypotheses 64: 694-698 ·
heart disease	Valen et al. (2001) Journal of the American College of Cardiology 38: 307-314

2017201593 08 Mar 2017

chronic heart failure	Frantz et al. (2003) Cardiovascular Research 57: 749-756 Gong et al. (2007) International Journal of Clinical Practice 61: 611-621
ischemia/reperfusion	Toledo-Pereyra et al. (2004) Experimental and Clincal Transplantation 2: 174-177 Nichols (2004) Drug News and Perspectives 17: 99-104 Ridder & Schwaninger (2008) Neuroscience 15 8(3):9951006
stroke	Herrmann et al. (2005) Nature Medicine 11: 13322-1329
cerebral aneurysm	Aoki et al. (2007) Circulation 116 2830-2840 Aoki et al. (2009) Arteriosclerosis, Thrombosis, and Vascular Biology 29:1080-1086
angina pectoris	Ritchie (1998) Circulation 98: 1707-1713
pulmonary disease	Christman et al. (2000) Chest 117: 1482-1487
cystic fibrosis	Bodas & Vij, Discovery Medicine, 9(47):346-356, April 2010 Pollard et al. (2005) Proteomics 5: 2210-2216 Carrabino et al. (2006) Journal of Cystic Fibrosis 5: 113119 Rottner et al. (2007) FASEB Journal 21: 2939-2948
acid-induced lung injury	Madjdpour et al. (2003) Anesthesiology 99: 1323-1332
pulmonary hypertension	Sawada et al. (2007) Chest 132: 1265-1274
chronic obstructive pulmonary disease (COPD)	Barnes (2002) Nature Reviews Drug Discovery 1: 437-446 Rahman & Kilty (2006) Current Drug Targets 7: 707-720
adult respiratory distress syndrome	Schwartz et al., Crit Care Med 1996;24:1285-1292.
hyaline membrane disease	Cheah et al. (2005) Pediatric Research 57: 616-623
kidney disease	Guijarro & Egido (2001) Kidney International 59: 415-424 Camici (2007) Medical Hypotheses 68: 900-905 Guzik & Harrison (2007) Circulation Research 101: 227229
glomerular disease	Zheng et al. (2005) Virchows Archiv 448: 172-183
alcoholic liver disease	Zima & Kalousova (2005) Alcoholism, Clinical and Experimental Research 29 (Supplement): 110S-115S
leptospirosis renal disease	Yang et al. (2001) Nephrology Dialysis Transplantation 16 Suppl 5: 73-77

2017201593 08 Mar 2017

gut diseases	Neurath et al. (1998) Gut 43: 856-860
peritoneal endometriosis	Gonzalez-Ramos et al. (2007) Molecular Human Reproduction 13(7):503-509
skin diseases	Bell et al. (2003) Cell Signaling 15: 1-7
nasal sinusitis	Xu et al. (2006) Neurochemistry Research 31: 1263-1269
anhidrotic ecodermal dysplasia-id	Puel et al. (2005) Journal of Endotoxin Research 11: 220224
Behcet's disease	Todaro et al. (2005) Arthritis and Rheumatism 52: 21792191
incontinentia pigmenti	Courtois & Israel (2000) Sci STKE Nov 14 (58): PEI
tuberculosis	Zea et al. (2006) Journal of Infectious Diseases 194: 13851393
asthma	Barnes, Am J Respir Crit Care Med 1996;154:S21-S27 Stacey et al., Biochem Biophys Res Commun 1997;236:522-526 Pahl & Szelenyi (2002) Inflammation Research 51: 273282
arthritis	Roshak et al. (2002) Current Opinion in Pharmacology 2: 316-321 Roman-Blas & Jimenez (2006) Osteoarthritis and Cartilage 14 839-848 Aud & Peng (2006) Nature Clinical Practice: Rheumatology 2: 434-442 Okamoto (2006) Endocrine, Metabolic & Immune Disorders: Drug Targets 6: 359-372
rheumatoid arthritis	Handel et al., Arthritis Rheum 1995;38:1762-1770 Sakurada et al., Int Immunol 1996;8:1483-1493 Roshak et al., J Biol Chem 1996;271:31496- 31501.
Crohn's disease	Pena & Penate (2002) Revista Espanola d Enfermedades Digestivas 94: 351-360
colitis	Chen et al. (2005) World Journal of Gastroenterology 11: 1508-1514
ulcerative colitis	Barnes & Karin, NEJM 336, 1066-71, 1997
ocular allergy	Bielory et al. (2002) Current Opinion in Allergy and Clinical Immunology 2: 435-445
glaucoma	Zhou et al. (2005) Journal of Biological Chemistry 280: 31240-31248
appendicitis	Pennington et al. (2000) American Surgery 66: 914-918

2017201593 08 Mar 2017

Paget's disease	Lin et al. (2007) Journal of Dermatological Science 45: 187-192
pancreatitis	Weber & Adler (2001) Pancreatology 1: 356-362 Gray et al. (2006) Pancreas 33: 260-267
periodonitis	Nichols et al. (2001) Annals of Periodontology 6: 20-29 ; Ambili et al. (2005) Journal of Periodontology 76: 11481153
endometriosis	Guo (2006) Gynecology and Obstetrics Investigations 63: 71-97 Celik et al. (2008) Human Reproduction 23: 2458-2465
inflammatory bowel disease	Neurath et al., Nature Med 1996;2:998-1004 Dijkstra et al. (2002) Scandinavian Journal of Gastroenterology Suppiemerit 236: 37-41 Atreya et al. (2008) Journal of Internal Medicine 263: 591 596
inflammatory lung disease	Park & Christman (2006) Current Drug Targets 7: 661-668
sepsis	Wratten et al. (2001) Contributions to Nephrology 132: 400-414
septic shock	Mukaida et al., J Leukoc Res 1996;59:145-151 Bohrer et al., J Clin Invest 1997;100:972-985.
silica-induced diseases	Chen & Shi 2002) Molecular and Cellular Biochemistry 234-235: 169-176 .
sleep apnea	Lavie (2003) Sleep Medicine Review 7: 35-51
viral infection (e.g., rhinovirus, influenzavirus)	Barnes & Karin, NEJM 336, 1066-71, 1997
aids (HIV-1)	Hiscott et al. (2001) Journal of Clinical Investigation 107: 143-151
allograft rejection	Lee & Burckart, J. Clin. Pharmacol. 38, 981-93, 1998 Bach et al., Nature Med 13, 944-48, 1997A Bach et al., Transplant Proc 29, 56-58, 1997B
autoimmunity	Hayashi & Faustman (2002) Diabetes Technology & Therapeutics 2: 415-428
antiphospholipid syndrome	Lopez-Pedrera et al. (2005) Arthritis and Rheumatism 54: 301-311

2017201593 08 Mar 2017

lupus	Kammer & Tsokos (2002) Current Directions in Autoimmunity 5: 131-150
lupus nephritis	Zheng et al. (2006) Human Pathology 37: 637-647
chronic disease syndrome	Maes et al. (2007) Neuro Endocinol Letters 28: 456-462
familial Mediterranean fever	Onen (2006) Rheumatology International 26: 489-496
hereditary periodic fever syndrome	Jeru et al. (2008) Proceedings of the National Academy of Sciences USA 105, 1614-1619
psychosocial stress diseases	Bierhaus et al. (2004) Pediatic Nephrology 19: 1189-1191
neuropathological diseases	Cechetto (2001) Progress in Brain Research 132: 391-404
familial amyloidotic polyneuropathy	Mazzeo et al. (2004) Archives of Neurology 61: 10971102
traumatic brain injury	Hang et al. (2005) World Journal of Gastroenterology 11: 1149-1154
spinal cord injury	Brambilla et al. (2005) Journal of Experimental Medicine 202: 145-156
Parkinson’s disease	Soos et al. (2004) Neuroreport 15 1715-1718
multiple sclerosis	Satoh et al. (2007) Neuroscience Letters 422: 30-33
rheumatic disease	Okamoto T (2006) Endocrine, Metabolic & Immune Disorders: Drug Targets 6: 359-372
Alzheimer’s disease	Mattson & Camandola (2001) Journal of Clinical Investigation 107: 247-254 Kaltschmidt et al., Proc. Natl. Acad. Sci. USA 94:26422647, 1997 Lukiw, J. Biol. Chem. 2008 Nov 14;283(46):31315-22. Epub 2008 Sep 18
amyotrophic lateral sclerosis	Xu et al. (2006) Neurochemistry Research 31: 1263-1269
Huntington’s disease	Khoshnan et al. (2004) Journal of Neuroscience 24: 79998008
retinal disease	Kitaoka et al. (2004) Brain Research Molecular Brain Research 131: 8-16
cataracts	Yang et al. (2006) Chinese Journal of Traumatology 9: 8690 .
hearing loss	Merchant et al. (2005) Otology and Neurotology 26: 151160
cancer	Gilmore et al. (2002) Cancer Letters 181: 1-9

2017201593 08 Mar 2017

solid tumors (generally)	Pacifico & Leonardi (2006) Biochemical Pharmacology 72: 1142-1152
breast cancer	akshatri et al. (1997) Molecular and Cellular Biology 17: 3629-3639
cervical cancer	Nair et al. (2003) Oncogene 22: 50-58
ovarian cancer	Lin et al., Clin. Cancer Res. 13, 3423-30, 2007 Samanta et al., J. Biol. Chem. 279, 7576-83, 2004
adenocarcinoma	Dejardin et al. (1999) Oncogene 18: 2567-2577
vulvar cancer	Seppanen & Vihko (2000) Immunology Letters 74: 103109
prostate cancer	Huang et al. (2001) Oncogene 20: 4188-4197
kidney cancer	Oya et al. (2001) Oncogene 20: 3888-3896 Oya et al. (2003) Carcinogenesis 24: 377-384
bladder cancer	Horiguchi et al. (2003) Expert Reviews in Anticancer Therapy 3: 793-798
lung cancer	Tichelaar et al. (2004) Chest 125: 153S
mesothelioma	Bertino et al. (2007) International Journal of Cancer 121: 2766-2774
non small-cell lung cancer	Zhang et al. (2006) Annals of Thoracic Surgery 82 243248 .
liver cancer	Tai et al. (2000) Cancer 89: 2274-2281
pancreatic cancer	Wang et al. (1999) Clinical Cancer Research 5: 119-127 Zhang & Rigas (2006) International Journal of Oncology 29: 185-192
esophageal/gastric cancer	Sutter et al. (2004) Onkologie 27: 17-21 Jackson & Evers (2006) Cancer Treatment and Research 130: 39-65
laryngeal cancer	Zhu et al. (2004) Lin Chuang Er Bi Yan Hou Ke Za Zhi 18: 745-6, 766
stomach cancer	Sasaki et al. (2001) Clinical Cancer Research 7: 4136-4142
colon cancer	Lind et al. (2001) Surgery 130: 363-369
thyroid cancer	Visconti et al. (1997) Oncogene 15: 1987-1994

2017201593 08 Mar 2017

parathyroid cancer	Corbetta et al. (2005) Endocrine Related Cancer 12: 929937
melanoma	Yang & Richmond (2001) Cancer Research 61; 4901-4909 Amiri KI and Richmond A (2005) Cancer Metastasis Reviews 24: 301-313
squamous cell carcinoma	Loercher et al. (2004) Cancer Research 64: 6511-6523
head and neck cancer	Ondrey et al. (1999) Molecular Carcinogenesis 26: 119- 129 Jackson-Bemitsas et al. (2007) Oncogene 26: 1385-1397
endometrial (uterine) cancer	Pallares et al. (2004) Journal of Pathology 204: 595-577
cylindromatosis	Kovalenko et al. (2003) Nature 424: 801-805
trichoepithelioma	Almeida et al. (2008) Dermatol. 127, 587-93
Hilar cholangiocarcinoma	Chen et al. (2005) World Journal of Gastroenterology 11 : 726-728
oral carcinoma	Nakayama et al. (2001) Cancer 92: 3037-3044 Ruan et al. (2007) Phytotherapy Research 22, 407-15
astrocytoma/gl ioblastoma	Hayashi et al. (2001) Neurologia Medico-Chirufica 41: 187-195 Smith et al. (2007) Molecular and Cellular Biochemistry 307:1-2, 141-147
neuroblastoma	Bian et al. (2002) Journal of Biological Chemistry 277: 42144-42150
glioblastoma	Raychaudhuri et al. (2007) Journal of Neurooncology 85: 39-47
Hodgkin’s lymphoma	Bargou et al. (1996) Blood 87: 4340-4347 Bargou et al. (1997) Journal of Clinical Investigation 100: 2961-2969
acute lymphoblastic leukemia	Kordes et al. (2000) Leukemia 14: 399-402
acute myelogenous leukemia	Guzman et al. (2001) Blood 98: 2301-2307
acute T-cell leukemia (+/HTLV-1)	Arima & Tei (2001) Leukemia and Lymphoma 40: 267278
acute non-lymphocytic leukemia	Lei & Zhao (2007) Zhongguo Shi Yan Xue Za Zhi 15 253257

2017201593 08 Mar 2017

chronic lymphocytic leukemia	Furman et al. (2000) Journal of Immunology 164: 22002206
Burkitts lymphoma (EBV)	Knecht et al. (2001) Oncology 60: 289-302
mantle cell lymphoma	Martinez (2003) Cancer Research 63: 8226-8232
myelodysplastic syndrome	Fabre et al. (2007) Oncogene 26: 4071-4083
multiple myeloma	Gilmore (2007) Cancer Cell 12 95-97 Berenson et al.(2001) Seminars in Oncology 28: 626-633
diffuse large b-cell lymphoma	Davis et al. (2001) Journal of Experimental Medicine 194: 1861-1874
MALT lymphoma	Sagaert et al. (2007) Leukemia 21: 389-396
Waldenstrom macroglobulinemia	Leleu et al. (2008) Blood 111:5068-5077
osteoporosis	Ray & Cohn, J. Clin. Invest. 104, 985-993, 1999; Christman et al., Chest 117, 1482-1487, 2000

Table 2. Dosing solutions

Test compound	Percentage content of anatabine or nicotine base	Dose level (mg/kg)	Test compound concentration(total) (mg/mL)	Test compound concentration (base) (mg/mL)	Injection volume (mL/kg)
Anatabine	41.6	0.10	0.048	0.020	5
Anatabine	41.6	0.75	0.36	0.15	5
Anatabine	41.6	1.0	0.48	0.20	5
Nicotine	35.1	0.4	0.23	0.081	5

2017201593 08 Mar 2017

Table 3. Phase I

Test compound	Route	Dose level (mg/kg)	Number of animals (M/F)	Collection times (minutes)3
Anatabine	i.v.	0.10	3/3	15, 30, 60, 90, 120, 240,360, 480,1440
Anatabine	i.v.	0.75	3/3	15, 30, 60, 90, 120, 240,360, 480,1440
Anatabine	i.v.	1.0	3/3	15,30, 60, 90, 120, 240, 360, 480,1440
Nicotine	i.v.	0.4 _	3/3 L	15,30, 60, 90, 120, 240,360, 480,1440

^a Plasma samples were collected at all time points,. Brain tissue was collected at 1440 minutes.

Table 4. Phase II

Test compound	Route	Dose level (mg/kg)	Number of animals per time point (M/F)	Collection times (minutes)3
Anatabine	i.v.	0.10	3/3	30, 360
Anatabine	i.v.	0.75	3/3	30, 360
Anatabine	i.v.	1.0	3/3	30, 360
Nicotine	i.v.	0.4	3/3	30, 360

^a Plasma samples and brain tissue were collected at all time points.

2017201593 08 Mar 2017

Table 5. Calibration Curve Concentrations

nominal concentration (ng/mL)	stock concentration (pg/mL)
5000	250
1667	83.3
555.5	27.8
185.2	9.3
61.7	3.1
20.6	1.0
6.9	0.34
2.3	0.11
0.76	0.038
0.25	0013

Table 6. LC/MS/MS ionization conditions and identity of parent and product ions

.Compound	MW	Polarization	Precursor m/z	Product m/z	Collision energy (V)
Anatabine	160.2	Positive	161.1	115.1	28
Nicotine	162.3	Positive	163.1	117.1	28
(+/-)-nicotine-3'-d3	165.25	Positive	166.1	118	28
(R,S)-Antabine-2,4,5,6-d4	164.24	Positive	165.1	148.1	20

Table 7. Limits of Detection and Calibration Curves

Sample	Limit of Detection (LOD) (ng/mL)	Lower Limit of Quantitation (LLQ) (ng/mL)	Upper Limit of Quantitation (ULQ) (ng/mL)
Anatabine in rat plasma	0.76	2.3	5000
Anatabine in rat brain	0.76	2.3	5000

2017201593 08 Mar 2017

Nicotine in rat plasma	0.76	2.3	5000
Nicotine in rat brain	0.76	2.3	5000

Table 8. Recovery from Plasma

	% Recovery at Given Concentrations
Sample	2.3 ng/mL	62 ng/mL	1667 ng/mL
Anatabine in rat plasma	74	96	NDa
Anatabine in rat brain	ND	96	90
Nicotine in rat plasma	ND	105	104
Nicotine in rat brain	ND	78	84

^aND - not determined; two points per condition were evaluated for measuring recovery.

Table 9. Dosing Solution Analysis

Compound	Dose (mg/kg)	Expected Concentration (mg/mL)	Actual Concentration (mg/mL)	Actual Concentration relative to Expected (%)
Nicotine	0.4	0.081	0.096	118.5
Nicotine	QA	0.081	0.096	118.5
Anatabine	0.1	0.02	0.014	70
Anatabine	0.75	0.15	0.135	90
Anatabine	1	0.2	0.177	88.5
Anatabine	0.1	0.02	0.015	75
Anatabine	0.75	0.15	0.133	88.7
Anatabine	1	0.2	0.162	81

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Anatabine	1.0 mg/kg	Female	346.0 + 56.7	6.0 + 2.8	<LLQ
Male	323.0 ± 35.9	5.0 + 2.6	c> -H cN
Anatabine	0.75 mg/kg	Female	314.7 + 67.9	21.0 + 6.2	<LLQ
Male	276.0 + 37.0	21.3 + 5.1	<LLQ
Anatabine	0.1 mg/kg	Female	29.7 + 4.5	3.7+1.5	2.0
Male	20.0 + 1.4	3.0	3.0
Nicotine	0.4 mg/kg	Female	120.0 + 3.0	5.0	7.0
Male	94.3 ± 20.6	6.0+1.4	CO CN -H m so
Treatment Group	Time (hr)	0.5		24

2017201593 08 Mar 2017

Table 13A.

Rat PK tv. dose -Brain Collection

CcwpaQDd: Anitibcx, Nxoa [>»»: 5 taL'kc

Roue: i.v.. PBS____________________

	24 hr
Crapnd	Rai	B.W.ta)	volume (mL)	time	rime	Brain wi(k)	Volume (nd.)
I	A		1.25	8:17	8:17	1.74	1.74
MALE	B	247	1.23	8:19	8:19	1.88	1.88
Aaauba· - OJ taj/kg	C	238	1J0	8:21	8:2 ί	185	1.80
' :	A	205	1.03	8:23	8:23 .	1.68	1.68
.FEMALE	B	2Π	LOS	8:25	8:25	1.83	1.83
Awubae 0.1 «i/k5	C	202	LOO	8:27	8:27	1 ..65	1.66
J	A	255	1.28	8:29	8:29	1.90	1.90
MALE	B	225	1.13	8:31	8;3l	1.32	1.82
¢.75 sng’i |	C	242	1.20	8:33	8:33	ι 1.84	1.84
4	A	204	LDJ	8:35	8:35’	1.82	/-1.82
FEMALE	B	205	1.03	8:37	S:37	1182	L82
Aaintna» 0 75 tag k|	C	210	1.05	8:39	8:39	1.71	1.71
$	A	242	1.20	8:41	8:41	1.83	1.83
MALE	B	251	1.25	8:43	8:43	1.35	1.85
Imnhx» 1.0 mg'lg	C	246	1.23	S:4S	8:45	l 90	1.90
6	A	213	1.08	’ 8:4? ’	8:47	' 1.95	.1.95
FEMALE	B	215	1.10	3.49	8:49	1.75	1.75
* Axuilua*	C	219	l.io	8:51	851	1.91	1.91
7	A	242	1.20	8-J4	8:54	2.03	2.03
MALE	B	241	1.20	8:56	8:56	198	1.98
Νχαπη 0.4iarY«	C	252	1.25	8:53	8:58	1.86	1.86
8	A	219	1.10	9:00	9:00	1.82	. 1.82 ' ‘
FEMALE	B	215	1.08	9:02	9Λ2	1.87	1.87
Nit Kia· 0.4 ca U	,c	220	1.10	9104	9104 ’	1.87	1.87 .
Court d Male	1	N7A	K/A	ΝΆ	N7A	1.83	1.83
X	NVA	N/A	N/A	N/A	154	1.94
	3	N/A	N/A	N/A	N/A	1.82	1.82

2017201593 08 Mar 2017

Table 13B. Animal Weights and Dosing Times

Rut PK tv. dose -Brain Collection

Dose: 5 mL/kg Compound: Anatabine, Nicotine

Route t Lv„ PBS_______________

	0.5 hour
Cmpnd	Rat	B.W. (gj	volume (mL)	lime	limo	Brain wt (g)	Volume (mL)
1	D	264	1.30	12:38	13:08	1.58	1.58
MALE	E	270	1.35	13:41	13:11	1.54	154
Anatabine 0.1 tn^'kp	F	2S2	1.26	12:45	13:15	169	169
2	D	220	1.10	12:46	13 16	178-	1 78
FEMALE	E	206	I.C3	12:50	13:20	1.54	1.54
Anatahhic 0.1 rqg/k#	F	214 ’	1.08	12:52	13 22	1.69	169
3	D	259	1.30	12:56	13:26	1.70	1.70
MALE	E	266	1 33	12:58	13:28	L66	166
Anatah'ne 0,7$ mjj'kg	F	364	1.33	13:03	13:33	1.46	146
4	D	208	1.05	13:05	13:35	LSI	LSI
FEMALE	E	219	L10	13:09	13:39	'-'i '1.84	1.84
AnmaLttv	F	223	LB	13:12	13:42	1.69	169
$	r>	276	1.38	13:16	13:46	2.00	2.1X1
MALE	E	25*	125	13:19	13:49	1.84	1.84
Anaatdiw 1.0	F	255	1.28	13:22	13:52	1.68	1.68
6	D	212	LOS	13:25	13:55	166	1.66
FEMALE	E	225-	LB	13:29	13:59	1 56	1 56
Anatafabtc 1.0	F	236	1:18	13:32	14.02	1.79	179
7	D	273	138	13:36	14:06	176	176
MALE	E	256	128	13.39	14.09	172	172
Nicotine 0.-1 nigtg	F	263	1.33	13:44	14:14	1.77	177
8	n	212	1.0.5	13:48	14:18	175	175
FEMALE	E	213	105	13:52	14:22	181	181
Nicotine 04 rwlg	F	231	1.15	13:59	14:29	190	1.90

Γο

ΓΜ

S ο

m ο

ΓΜ

ΓΟ ΓΜ

Concentration (ng/mL)	o	r-	102	un	<LLQ	r-	un	un	o\	Γ-	120
X
Ο
d ti
6 -S •Ξ o	un		un				TT			un	un
H Cu	©	o	o	Ό		kD		Γ4	C4	o	o
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C£	r*	r-	r*	Γ--	r*	r-	r-r	r*	r·*	00	00
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	rt	rt	rt	rt	rt	ci		cb	co	rt
	l.	k.	k.	u>		u		u		u	Im
3	CQ	03	cn	cn	cn	cn	CQ	cn	cn	cn	cn
	rt	rt	¢9	rt	OS	aS	irt	<0	rt	rt	rt
H	at	al	¢2	at	c2	c2	cd	at	¢2	c2	al
	00	00	00	00	00	00	00		00	00	oo
	ine g/k	ine g/k	ine g/k	.£	ine g/k	ine g/k	.S oi)	ine g/k	ine g/k	ine g/k	.ii
cu §	o E	o 6	ο E	’ o 2	ο E	© E	ο E	5 E	ο E	O fa	ο E
O 2	.2 rt-	.2 rt	.2 rt	.2 rt	.2 rt	.2 rt	.2 rt	.2 rt	.2 rt	.2 rt	.2 rt
Q O	Z o	Z o	Z o	Z o	Z o	Z o	o	Z o	Z o	Z o	Z o
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□	<u	ω	<υ	Q	0)	Q	o	o	<υ	<0	o
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C	o	o	o	o	o	o	o	o	o	o	o
	Q	Q	o	Q	o	Q	Q	o	Q	Q	Q
o
u	z	z	z	z	z	z	z	z	z	z	z

2017201593 08 Mar 2017

Concentration (ng/mL)

123

<LLQ

<LLQ

<LLQ

Γ*·

<LLQ

<LLQ

0s.

CM

<LLQ

Time Point (hr)

0.5

80

8©

80

24

24

24

0.5

1 0.5

0.5

80

80

Rat ID

8F

8G

K OO

00

00

CQ 00

o 00

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w

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Tissue

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Rat Brain

Dose Group

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Anatabine 0.1 mg/kg

Anatabine 0.1 mg/kg

Anatabine 0.1 mg/kg

Anatabine 0.1 mg/kg

Anatabine 0.1 mg/kg

Compound

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Anatabine

Anatabine

Anatabine

Anatabine

Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)

<LLQ

m

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34

25

30

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2017201593 08 Mar 2017

Concentration (ng/mL)	<LLQ	266	317	245	r*-	27	20	<LLQ	<y V
Time Point (hr)	24	0.5	0.5	0.5	vo	VO	VO	24	24
Rat ID	2C	3D	3E	EXl	o CO	3H	m		3B
Tissue	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain
Dose Group	Anatabine 0.1 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine ____________________________________________________________________________________________________________________________________________________________________________________________________________________________________1

2017201593 08 Mar 2017

Concentration (ng/mL)	<LLQ	393	272	279	Ό	28	o	<LLQ
Time Point (hr)	24	0.5	0.5	0.5	Ό		k©	CM
Rat ID	o	4D	4E	4F	4G	4H	5	4A
Tissue	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)	<LLQ	<LLQ	349	338	282	cn		oo	cn	CN	<LLQ
Time Point (hr)	24	24	m o	0.5	0.5	Ό	kO	kO	24	24	24
Rat ID	4B	4C	5D	5E	5F	5G	5H	n	5A	5B	5C
Tissue	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	1 Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)

362

1 393 .

283

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194

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2017201593 08 Mar 2017

Concentration (ng/mL)

CH CN

00

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187

oo

157

72

67

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Time Point (hr)

0.5

0.5

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Rat ID

7A

7B

7C

7D

7E

r-

7A

7B

7C

7A ·

7B

7C

Tissue

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Dose Group

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Compound

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

2017201593 08 Mar 2017

Concentration (ng/mL)

CM

32

CM

<LLQ

<LLQ

<LLQ

<LLQ

1 <LLQ

<LLQ

<LLQ

<LLQ

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Time Point (hr)

CM

CM

CM

Μ-

80

80

Ό

Ό

80

80

1 Rat ID

7 A

7B

7C

7A

7B

7C

7A

7B

7C

7G

7H

1------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Tissue

Rat .. Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

1 Dose Group

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Compound

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

2017201593 08 Mar 2017

Concentration (ng/mL)

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

175

145

184

95

125

Time Point (hr)

OO

OO

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24

24

24

0.25

0.25

0.25

0.5

0.5 i

0.5

Rat ID

7A

7B

7C

7A

7B

7C

< OO

CQ oo

o oo

00

8B

o oo

Tissue

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Dose Group

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Compound

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

2017201593 08 Mar 2017

Concentration (ng/mL)

180

157

160

67

72

107

49

37

64

25

24

46

Time Point (hr)

0.5 1

0.5

0.5

IT)

in

in

CM

CM

CM

Rat ID

8D

8E

8F

00

1 8B i

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8B

8C

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00

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Tissue

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Dose Group

1 Nicotine 0.4 mg/kg j

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Compound

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

2017201593 08 Mar 2017

Concentration (ng/mL)

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

<LLQ

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Time Point (hr)

kO

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00

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Rat ID

00

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Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Rat Plasma

Dose Group

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Nicotine 0.4 mg/kg

Compound

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

Nicotine

2017201593 08 Mar 2017

Concentration (ng/mL)

<LLQ

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24

20

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30

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2017201593 08 Mar 2017

Concentration (ng/mL)

o

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2017201593 08 Mar 2017

Concentration (ng/mL)

<LLQ

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Rat Plas

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2017201593 08 Mar 2017

Concentration (ng/mL)

m

30

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2017201593 08 Mar 2017

Concentration (ng/mL)

CN

CN

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m

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ra

ra

ra

ra

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ra

ra

ra

ra

ra

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2017201593 08 Mar 2017

Concentration (ng/mL)	<LLQ	co	<LLQ	<LLQ	<LLQ	<LLQ	<LLQ	<LLQ	<LLQ	216	162
Time Point (hr)	kD	kO	kO	oo	oo	QO	24	24	24	0.25	0.25
Rat ID	2A	2B	2C	2A	CQ CN	2C	2A	2B	u CN	3A	m CO
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)	m CM CM	176	176	190	342	271	292	153
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ime oint	m CM				wn	ι/Ί	un
H cu	ό	o	o	O	o	O	o
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cd	u	<	CQ	o	Q	id	tx	<
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(D =3	cd ,	cd	cd	cd	cd	cd	cd	cd
ε	ε	ε	ε	ε	ε	ε	ε
Tiss	Rat Plas	Rat Plas	Rat Plas	Rat Plas	Rat Plas	1 Rat Plas	Rat Plas	Rat Plas
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CL	·§ op	·§ op	·§ gp	§ op	op	§ op	S SP	§ op
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Q O	< © ε	< ο ε	< © ε	< © ε	< © ε	< © ε	< © ε	< © ε
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2017201593 08 Mar 2017

Concentration (ng/mL)	146	156	120	124	136	co r*		104
ime oint			in	in	in
H CL,						CN	CN	CN
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Q 3	rt	rt	rt	rt	rt	rt	rt	rt
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CL	=p	rt sp	·§ SP	5 SP	•rt «>	rt M	rt SP	rt SP
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Q C	< ο E	< ο E	< ο E	< <=> E	< ο E	< <=> E	< ο E	< ο E
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3	3	c	C	3	3	3	3	3
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2017201593 08 Mar 2017

Concentration (ng/mL)	29	36	Os en	un	Tt	cn	m	en
Time Point (hr)				o	O		Ό
Rat ID	3A	CO cn	3C	< m	3B	U cn	O cn	X cn
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)

20

QO

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2017201593 08 Mar 2017

Concentration (ng/mL)	226	207	166	229	175	307	359	396
Time Point (hr)	0.25	0.25	0.5	0.5	0.5	0.5	0.5	0.5
Rat ID	4B	4C	4A	4B	4C	Q		4F
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)	146	207	165	136	134	150	68	cn
Time Point (hr)	-			in	in	in	CN	CN
Rat ID	4A	4B		4A	1 4B	4C	4A	4B
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)	97	45	50	'Sj-		23	OO	UY
Time Point (hr)	CM				kO	Ό	so	kO
Rat ID	4C	4A	.. CQ St	4C	4A	4B .	4C	4G
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 - mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)	38	kO	o	CM		<LLQ	<LLQ	1 <LLQ	296
Time Point (hr)	kD	kO	00	OO	OO	24	24	1 24	0.25
Rat ID	X	5	4A	4B	4C	4A	4B	u	5A
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	i------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	1 Anatabine 0.75 mg/kg	Anatabine 0.75 img/kg	Anatabine 0.75 mg/kg	1--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Anatabine 0.75 mg/kg	Anatabine 1.0 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

2017201593 08 Mar 2017

Concentration (ng/mL)

291

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2017201593 08 Mar 2017

Concentration (ng/mL)

148

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2017201593 08 Mar 2017

Concentration (ng/mL)

1 38

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293

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2017201593 08 Mar 2017

Concentration (ng/mL)

253

236

347

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218

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2017201593 08 Mar 2017

Concentration (ng/mL)

170 1

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2017201593 08 Mar 2017

Concentration (ng/mL)	o\		<LLQ	<LLQ	<LLQ
ime oint (hr)
		tT	’iT
r* 0-	00	00	CN	CN	CN
Q
ee	CQ	u	<	CQ	u
ai	X	X	X	X	X
ue	cQ	cQ		cQ	CQ
E	E	E	E	E
1 Tiss	Rat Plas	Rat Plas	Rat Plas	Rat Plas	Rat Plas
	Ο ©Λ	on		<υ on	i> on
CL	atabin mg/k	atabin mg/k	atabin mg/k	atabin mg/k	atabin mg/k
o £	C O	c o	c o	G O	c o
Q O	< -	< -	< -	< -	< -
	<υ	o	,o	Q	<υ
5	c	G	c	C	c
O	X	X	X	X	X
CL	es	CQ	cs	CQ	CQ
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Γo CN

sem	7.9	0.7	0				2.8
JD cO g > § 44 u- Ί2 -c	19.3	CN	OS				6.2
G cO ο) II co C	80	cn	''t				tn
· TJ 2 c § <S § m •S * « ϋ - « £ c ° 2 ma E S T\ 0 5 5 > > « Ε <_) 4 B< < -i Q, C JO	107.2	in	6.5				25.8
s ω IZI -H	O\	0	1.3		ND	ND	0
iSTDEV	20.6	1.4	2.3	3.0	ND	ND
Ό 2 «- ”c $2 0 <2 w _ Ξ U - =2 <2 <U . P P O' cu S 00-E 5 c -2 bO E Ο <ζ r*>. g	94.3	6.0	6.3	120.0	5.0	7.0	20.0
Ίο S Z 11	cn	CM	cn	cn		—	CM
Time Point (hr)	0.5	80	24	0.5	80	24	0.5
Sex	male	male	male	female	female	female	male
Tissue	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain
Dose Group	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Anatabine 0.1 mg/kg
Compound	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Anatabine

100

2017201593 08 Mar 2017

sem	0.6	0.5				21.8	CM	ND
JD cd 0 « £ S -o	cn	rό				53.3	<n	ND
4) II rc c	ΤΓ	CM				0		0
3 T3 CoS c c O * g « u = « <2 E — g 6b~§ « 00.S 0 « 5 > λ g U Z U. < 0 Q. C X>	3.5	2.5				295.3	21.2	<LLQ
s id oo -H	ND	ND	2.6	6‘0	ND	21.4	3.0	ND	39.2
> w Q H 00 -H	ND	ND	4.5	in	ND	37.0	in	ND	67.9
e or Female Avg. Cone, (ng/ml for plasma and ng/g for brain)	3.0	0 cn	29.7	3.7	2.0	276.0	21.3	<LLQ	314.7
rc .2 z 11	—	—	cn	cn		cn	cn	0	cn
Time Point (hr)	$0	’dCM	0.5	kO	’’φ CM	0.5	'sO	C4	0.5
Sex	male	male	female	female	female	male	male	male	female
Tissue	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain
Dose Group	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

101

2017201593 08 Mar 2017

sem			00	—	0.5				8.5
JU re c			44.3	2.4	0.7				20.8
u re tu II 're c			kO		cd
. _ T3 y >- 32 2 32 o<2 « u •g § Ji O - * <2 _ E 32 E ob-M ί m.E o « 5 > g> « s (J X < 6 Q. C X			334.5	5.4	2.5				166.5
ω <Z) -H	3.6	ND	20.7	in	0.5	32.7	2.0	ND	14.8
±STDEV	6.2	ND	35.9	2.6	0.7	56.7	2.8	ND	25.7
e or Female Avg. Cone, (ng/ml for plasma and ng/g for brain)	21.0	<LLQ	323.0	5.0	2.5	346.0	6.0	<LLQ	165.0
*re · S Z II	m	o			Cd	m	Cd	©	cn
Time Point (hr)		^r rd	0.5		Cd	0.5	kO	Cd	0.25
Sex	female	female	male	male	male	female	female	female	male
Tissue	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Brain	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Nicotine 0.4 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Nicotine

102

2017201593 08 Mar 2017

sem	7.0	9.9	6.4	in	3.9	0.5	ND	ND!	ND
-2 05 E > £ -2 x	17.2	24.1	15.6	12.5	9.6	0.7	ND	ND	ND
u «3 D II — c 05	\©	S©	s©	s©		CM	©	o	o
Combined M Male and Female Avg. Cone, (ng/ml for plasma and ng/g for brain)	104.8	159.8	75.5	42.8	28.2	3.5	<LLQ	O J u w	<LLQ
s ω	Ch	20.0	in	4.3	3.7	ND	ND	ND	ND
±STDEV	20.6	34.6	2.6	7.4	6.4	ND	ND	ND	ND
¢5 O <2 Ε O — 2 <2 _ U. 60-55 M.S u > g> w Yh 2 o < i C. C Xi	99.3	154.0	69.0	35.7	24.7	<LLQ	<LLQ	<LLQ	<LLQ
w S Z ii	CO	CO	cc	cn	cn	©	o	©	©
Time Point (hr)	0.5	0.5		m	CM		s©	ΟΦ	24
Sex	male	male	male	male	male	male	male	male	male
0) 3 cn <Z) P	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg
Compound	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine

103

2017201593 08 Mar 2017

sem	37.1	18.8	31.7	15.3	10.9	6.5	0.4	in	VC
¢0 g > __ w Ί3	83.0	65.0	77.7 1	37.6	24.4	15.8	0.9	N	2.1
U <0 D II c	X	CM	X	X	in	X	in	CN	<N
ί o u. <U E E .P 6 = c8 © <_> — w ,o E g § ^n.E ο « g > J2 ^3) E O z u. < 5 a c x	tz 801	79.7	95.3	50.8	33.6	19.2	3.6	5.5	5.5
±SEM	OO	8.7	7.2	12.6	7.8	7.2	0.5	ND	ND
±STDEV	20.4	15.0	12.5	21.8	13.5	12.4	0.7	ND	ND
w 2 >- e 2 ο « “ ι- Ε U - <υ . S P u- > g> _E E o < Q. C X	168.0	110.3	165.7	82.0	50.0	31.7	3.5	<LLQ	<LLQ
CO S z II	rc	rc	rc	rc	m	rc	(N	o	o
Time Point (hr)	0.25	0.5	0.5		uc	C4		X	X
Sex	female	female	female	female	female	female	female	female	female
Tissue	Rat Plasma	Rat ( Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg
Compound	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine	Nicotine

104

2017201593 08 Mar 2017

sem	ND	ND		2.7	0.7	CN	1.2	m	CN
Έ g > o	ND	ND	4.2	9.4	X	5.2	2.8	3.6	2.6
u ¢0 ω II 75 c	—	o	X	CN	X	X	in	X	m
έ 2 - s « §<2 L •f § « o - « <2 E g § M.S o 5 S > g> 42 E O 4 fa < C ft C Λ	4.0	o j ν’	0 81	22.9	31.7	8.0	9.2	8.7	5.2
s W <Z) Ή	ND	ND	2.9	1.2	60	o\	0.5	60	0.0
iSTDEV	ND	ND	5.0	CN	in	3.2	0.7	in	0.0
e or Female Avg. Cone, (ng/ml for plasma and ng/g for brain)	<LLQ	<LLQ	19.3	15.3	31.7	12.3	6.5	6.3	3.0
λ S Z II	o	o	cn	m	cn	m	CN	cn	CN
Time Point (hr)	oo	ΓΝ	0.25	0.5	0.5		in	CN
Sex	female	female	male	male	male	male	male	male	male
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Nicotine 0.4 mg/kg	Nicotine 0.4 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg
Compound	Nicotine	Nicotine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

105

2017201593 08 Mar 2017

sem	90	0.9	ND	ND
_u C3 g > ο. Έ o	in	1.7	ND	ND
u- re 43 . II re c	x©	’d-	o	o
i y - c ω Ό E ,ο ΐ5 e c © ti— k. - si « U - π o ε δ ithsOl	4.3	4.5	<LLQ	<LLQ
s w co -H	ND	o	ND	ND	2.2	r-	rd	0.3	.0 »·Μ
iSTDEV	ND		ND	ND	3.8	3.0	2.1	0.6	1.7
32 ggc 2 o « s l. E U — « <g W · E E I? tu βο-E S M.S u > g> « ^t) δ o < o o. s x>	<LLQ	5.0	<LLQ	<LLQ	16.7	13.0	31.7	e'en	o
re IS Z II	o	m	o	o	cn	er	cn	en	er
Time Point (hr)		©	00	24	0.25	0.5	0.5	80
Sex	male	male	male	male	female	female	female	female	female
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg
Compound	Anatabine	Anatabine	a Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

106

2017201593 08 Mar 2017

sem							9.5	23.7	18.9
JU ra g > S « u< 2 TJ							23.4	82.2	46.4
3 rt <p II ”rt c							k©	CN	k©
z TJ — c 1= S 1? o <2 § ·= ra .2 <J — Λ <2 _ E 75 E bb-|L § w .2 a siΐIm							206.3	256.6	327.8
s ω C75 -H	cn	cn	o	ND	ND	ND	19.3	4.7	21.1
> ω £ co . -H	3.6	2.3	rt;	ND	ND	ND	33.4	00	36.5
u y <- ra o 2 Λ i_ E u - 2 £ <D . E E c U- bO>L S bO.S = > ™ « ab 2 o < 5 a c λ	o	6.7	4.0	3.0	<LLQ	<LLQ	200.3	180.7	301.7
rt S Z ii	cn	cn	CN	MM	o	o	cn	cn	en
Time Point (hr)	UY	CN	rt·	k©	oo	rt* CN	0.25	0.5	0.5
Sex	female	female	female	female	female	female	male	male	male
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.1 mg/kg	Anatabine 0,.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.1 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

107

2017201593 08 Mar 2017

sem	9.4	4.3	6.6	3.0	2.2	4.0	0.7	ND
fl ra % 1 _ 1/3	23.1	10.6	k©	7.3	7.8	9.7		ND
C ra o II ra c	k©	X©	k©	<©	CN	k©	X©	o
T3 g -O <= •2 π “ u - “i <2 o 5 § > 1> « E O Z fc < ό a c Λ	162.2	133.3	91.7	40.0	19.3	22.5	0ΌΙ	<LLQ
s ω CZ) -H	3.0	4.8	10.2	3.0	9Ό	4.7	o	ND	6.9
+STDEV	in	8.3	17.6	in	o	8.2		ND	Ox
« g 5 Έ « o ¢5 Λ u ε u -= 2 <θ <u . P P — U, W)<5 M.S j, > g> « E o < O Q. c x>	151.7	126.7	83.7	34.7	14.0	22.0	9.0	<LLQ	212.3
*ra S Z ll	cn	cn	cn	m	cn	cn	cn		cn
Time Point (hr)		m	(N		k©	k©	00	24	0.25
Sex	male	male	male	male	male	male	male	male	female
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

108

2017201593 08 Mar 2017

sem
Ί5 J 1
ϋ II ri c
O — ri u ό Ε .0 E c S O G- u. ·= ri o — * <2 E -g 1 “>!> ^·§ O 5 S > “ « bh 2 Q u. < o c. c x>
I i ±SEM	19.7	25.8	18.0	5.0	7.1	2.6	2.6	7.5	0.6
iSTDEV	34.1	44.7	31.2	8.7	12.2	4.5	4.5	13.0	o
e or Female Avg. Cone, (ng/ml for plasma and ng/g for brain)	0Ό61	354.0	172.7	140.0	99.7	45.3	18.3	23.0	o
03 S z ll	cn	cn	cn	cn	m	cn	cn	cn	cn
Time Point (hr)	0.5	0.5		in	CM	Tt	so	so	QQ
Sex	female	female	female	female	female	female	female	female	female
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
i Dose Group	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg	Anatabine 0.75 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

109

2017201593 08 Mar 2017

sem		5.5	CN	14.5	6.4	o	9.9	5.4	CN
ce E > 73		13.4	73.1	35.4	15.7	24.7	24.3	13.2
u. cd ο II Έ c		VO	CN	Ό	VO	VO	VO	VO	CN
· T> o θ Ί= 2 -g §<2 § _ •S <u 2 · Ε ε c ε ~ E 55 -5°·= o 5 S > I142 2 U u. < c- a c		287.2	319.1	383.5	231.3	178.3	143.3	51.5	24.3
S ω 00 -H	ND	8.0	7.8	24.6	OO	4.6	en	4.5	1/Ί
> ω Q H (Z> -H	ND	13.8	13.6	42.5	20.4	7.9	5.3	7.8	2.6
e or Female Avg. Cone, (ng/ml for plasma and ng/g for brain)	<LLQ	285.7	272.3	399.0	233.7	157.0	123.0	42.3	18.0
S Z II	o	m	en	cn	cn	cn	en	cn	cn
Time Point (hr)	’d CN	0.25	0.5	0.5			CN	'd-	kO
Sex	female	male	male	male	male	male	male	male	male
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 0.75 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg
1 Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

110

2017201593 08 Mar 2017

sem	CO	2.3	ND
_ω g > £ s	v©	5.8	ND
13 ο II re c	V©	V©	o
g “ C O 73 S ,O ffl r- C O U— »— 15 * O -= g <2 E =; E 1 _$p ·§ o * 5 > s O Z U. < O Q. c x>	25.0	12.7	<LLQ
s tL> (Z) -H	V©	0.9	ND	9.2	9.0	14.2	7.8	5.8	8.4
±STDEV	10.5	1.5	ND	15.9	15.5	24.6	13.5	0ΌΙ	14.6
u 2 *- c <S o «2 * u E Q - g <2 ϋ · Ε E c tu ao>. S oo.S 2 > g1 * SD ε ο < ο cl c x>	27.0	LL	<LLQ	288.7	237.0	368.0	229.0	199.7	163.7
re S Z II	CO	co	o	CO	co	CO	CO	CO	CO
Time Point (hr)	*©	00	st CM	0.25	0.5	0.5		uo	<s
Sex	male	male	male	female	female	female	female	female	female
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

in

2017201593 08 Mar 2017

sem	*
¢3 I 3 x>
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s ω GO -H	6.3	4.2	2.6	en	ND
±STDEV	0	7.2	4.6	2.3	ND
ω υ v. 2 2 ο <2 « ι- Ε U - - a> ρ C m-ι zps b ώ<5 οη.Ξ ^b Ε Ο <_> ft. g	60.7	29.3	23.0	r* P	<LLQ
CS S z 11	m	ΓΊ	en	m	©
Time Point (hr)			so	00	Xf CM
Sex	female	female	female	female	female
Tissue	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma	Rat Plasma
Dose Group	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg	Anatabine 1.0 mg/kg
Compound	Anatabine	Anatabine	Anatabine	Anatabine	Anatabine

112

2017201593 08 Mar 2017 co

C o •w

O co

CO

Injection volume (mL/kg)		tn	tn	tn
Test compound base concentration (mg/mL)	0.048	0.36	0.72	0.36
Dose level (mg/kg)	0Γ0	0.75	1.5	tn ro
Percentage content of anatabine or nicotine base	41.6	41.6	41.6	35.1
Test compound	Anatabine	Anatabine	Anatabine	Nicotine

s O

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Observations, body weight and food consumption frequency	Daily for 14 days	Daily for 14 days	Daily for 14 days	Daily for 14 days	Not applicable	Daily for 14 days
Number of animals (M/F)	! 5/5	5/5	5/5	5/5	5/5	tn m
Dose level (mg/kg)	0'0	0Γ0	0.75	<n	<n	0.75
Route	>	>	>	i.v.	>	>
Test compound	Vehicle	Anatabine	Anatabine	Anatabine	Nicotine	Nicotine1

113

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Tests performed/tissues collected	Hematocrit, Hemoglobin, MCH, MCH Concentration, MCV, RBC, Reticulocyte count, Platelet count, WBC, WBC differential, blood smear evaluation	A/G ratio (calculated), ALT, Albumin, Alkaline phosphatase, AST, Bilirubin, Calcium, Chloride, Cholesterol (total), Creatinine, Globulin (calculated), Glucose, Phosphorus (inorganic), Potassium, Sodium, Total protein, BUN	Activated partial thromboplastin time, Prothrombin time	Bilirubin, Blood, color and appearance, Glucose, Ketones, pH, Protein, Specific gravity, Urobilinogen, Volume	Adrenal glands', Brain, Heart, Kidneys, Liver, Lungs, Ovaries and Uterus, Pituitary gland1. Prostate gland, Spleen, Testes, Thymus, Thyroid and Parathyroid glands', section of small intestines
Parameter	Hematology	Clinical Chemistry	Coagulation	Urinalysis	Necropsy

114

2017201593 08 Mar 2017

Actual Concentration relative to Expected %	118.5	80	85	119.5
Actual Concentration (mg/mL)	960Ό	0.016	0.127	0.239
Expected Cone, (mg/ml)	0.081	0.02	0.15	0.2
X—z	0.4	o	0.75 i	X
Dosing Date	Jun 16	Jun 09	Jun 09	Jun 09
Compound	Nicotine	Anatabine	Anatabine	Anatabine

115

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' ρ (comparison to Vehicle)			o.ooi |	0.535 |	0.936 |	0.434 |	o.ooi |	0.504 |	0.098 |	00 Ct in o’
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Anatabine 1.5 mg/kg

Nicotine 0.75 mg/kg

120

Table 24. Hematology Parameters (descriptive statistics) (Part 2) by Treatment

Group and Gender

2017201593 08 Mar 2017

Treatment Group	Gender		NEUTROPHIL SEG %	LYMPHOCYTE %	MONOCYTE %	EOSINOPHIL %	BASOPHIL %
Vehicle	··*'*'* 'M '	n	5	5	5	5	5
fctriitJL ., ao . IT ίββ_____		WM·	raw
Std Dev	2.2	2.3	1.5	0.0	00
F	N	5	5	5	5	5
Mean	12.6	85.2	2.2	0.0	o.o
Std	2.6	2.9	0.4	0.0	0.0
Anatabine 0.1 mg/kg		n	5	5	5	5	5
sWeanSI	«92-0.VJ.	ο% ’	• ” 0 2‘·· '	[ (βίή Ί
Std Dev	3.6	2.9	1.0	0.4	0.0
Pa	nsb	ns	ns	ns	ns
F	n	5	5	5	5	5
-Mean	.- 80 .. ,.·	- 90.2	1.8	0.0	0.0 -
Std Dev	1.9	1.9	0.8	0.0	0.0
P	ns	ns	ns	ns	ns
Anatabine 0.75 mg/kg		n	5	5	5	5	5
'kill ,9b	. 88 8	-= '’1 4	i) (	IWoTHW
Std Dev	2.5	2.2	0.5	0.0	0.0
P	ns	ns	ns	ns	ns
F	n	5	5	5	5	5
Mean	8.6	90.6	+ >-0.8'* '	0 0	.00 <
Std Dev	2.3	2.6	0.8	0.0	0.0
P	ns	ns	ns	ns	ns
Anatabine 1.5 mg/kg	M ...	n	5	5	5	5	5
Mean ·.*;	-14 6 .	»4'£83.8#ΜϊΛ	- -μ ι (<·-
Std Dev	7.7	8.0	0.9	0.0	0.0
p	ns	ns	ns	ns	ns
Γ	n	5	5	5	5	5
Mean '	» -^12.6^ te	86.6 '	:· . - 0 3 .	-- i Oo - -	- ·· 00 ’
Std Dev	4.4	3.8	0.8	0.0	0.0
P	ns	ns	ns	ns	ns
Nicotine 0.75 mg/kg		n	4	4	4	4	4
	MWilS···	mHrs^wn·			ra·#
Std Dev	3.3	4.2	1.0	0.0	0.0
	P	ns	ns	ns	ns	ns
F	n	3	3	3	3	3 .
Mean	12.7	86.3	1 0	0.0	' 010’
Std Dev	2.1	3.1	1.0	0.0	0.0
2	ns	ns	ns	ns	ns

“ p. probability relative to Vehicle control ^b ns, not significant

121

Table 25. Coagulation Parameters (descriptive statistics) by Treatment Group and Gender

2017201593 08 Mar 2017

Treatment Group	Gender		ACTIVATED PARTIAL THROMBOPLASTIN TIME (seconds)	PROTHROMBIN TIME (seconds)
Vehicle	?/Μβϊ·	n	5	5
f Mean '	' · .· 32.0 !	12.9 ·-
Std Dev	2.2	0.5
F	n	5	5
Mean	35.4	12.7
Std Dev	4.1	0.3
Anatabine 0.1 mg/kg	M	n	5	5
Mean	‘ ·· ' ' 32.4 · ‘	12.8'?:SiWfS3?W:
Std Dev	3.3	0.7
p‘	. nsb	ns
F	n	5	5
Mean	29.4	12.1
Std Dev	3.4	0.4
P	ns	ns
Anatabine 0.75 mg/kg	M	n	5	5
Mean	38.7
Std Dev	9.1	2.6 .
P	ns	ns
F	n	5	5
Mean	33.3	14.1
Std Dev	2.8	5.5
P	ns	ns
Anatabine 1.5 mg/kg	M	n	5	5
Mean	30.6	' v ‘·. 13.2
Std Dev	3.1	0.4
P	ns	ns
F	n	5	5
Mean	32.5	13.3
Std Dev	2.9	0.2
P	ns	. ns
Nicotine 0.75 mg/kg	M.	n	4	4
Mean .·	- 16.3	13.6 '
Std Dev	1.1	0.6
P	<o.oor	ns
F	n	2	2
Mean	16.9	14.1
Std Dev	2.6	0.2
S.	0.002'	ns

p. probability relative to Vehicle control ^b ns. not significant ^c Mean within normal range

122

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126

Table 28A. Dosing Calculations and Body Weights, days 1-5 (anatabine)

2017201593 08 Mar 2017

			Dose	time	dav 1	day 2	dav 3	dav 4	day 5
		B.W.	volume	of	B. W.	B. W.	B. W.	B.W.	B. W.
Rat	M/F	(g)	(mL)	dosing	(g)	(g)	(g)	(g)	(g)

A-1	1 M	250	1.25	10:39	258.0	270.0	280.3	296.0	306.6
vehicle	2	M	228	1.15	10:41	238.0	251.0	257.8	273.7	286.0
5 mL/kg	3	M	235	1.18	10:43	241.0	250.0	260.2	278.0	287.0
A-2	4	M	224	1.13	10:44	230.0	239.0	247.6	262.6	267.0
	5	M	236	1.18	10:45	241.0	253.1	261.7	281.0	289.0
A-3	6	F	207	1.03	10:49	210.2	220.5	218.1 ’	224.2	v 227.0
	7	F	221	. 11	10:50	221.9 '	222.0	225.1	2-292¾	ij2&r7
	8	F	209	1.05	10:51	210.0	214.0	216.9		»220.0
A-4	9	F	201	1	10:51	200.0	200.1 '	205.0 '	i 21613	bbs-o
	10	F	211	1.05	.10:52	208.4	24 /5^0 *	• 216¾}
B-l	1	M	237	1.18	10:48	245.0	257.0	270.0	286.0	296.5
Anatabine	2	M	227	1.13	10:49	233.7	235.1	251.0	270.0	273.8
0.1 mg/kg	3	M	230	1.15	10:50	235.0	231.0	252.0	271.2	278.5
B-2	4	M	243	1.23	10:52	243.5	253.5	259.4	280.5	288.3
	5	M	235	1.18	10:55	239.9	247.3	256.6	273.7	286.6
B-3	6	F	226	1.13	10:56	223.0	223.9	230.8	r 2426X	^246.9^
	7	F	212	1.05	10:57	211.1	215.1	213.2	222A	irsi- y > 226.4.
	8	F	207	1.03	10:58	207.0	208.1	209.1	220¾	219.3?
B-4	9	F	205	1.03	10:58	201.0	208.8	209.1	21 Γ:4,?	.221.0 ’
	10	F	215 .	1.08 .	10:59	210.9 -	212.8	219.3.:	V- 224.74£	229.5 .

127

2017201593 08 Mar 2017

Dose day 1 day 2 day 3 day 4 day 4

B.W. volume B. ,W. B. W. B. W. B.W. B.W.

Group Rat M/F (g) (mL) time (g) (g) (g) (g) (g)

C-l	1	M ,	239	1.2	10:58	242.7	248.9	261.1	275.7	282.3
Anatabine	2	M	241	1.2	11:00	251.4	259.7	269.1	286.0	287.6
0.75 mg/kg	3	M	229	1.15	11:02	231.9	240.0	252.0	268.0	269.9
C-2	4	M	223	1.13	11:04	226.8	232.1	243.0	256.3	262.0
	5	M	240	1.2	11:06	239.8	247.2	256.1	271.2	275.7
C-3	6	F	214	1.08	11:01	208.9	206.2	214.0	230.0	225.4
	7	F	206	1.03	11:02	207.7	210.0	214.0	214.7	219.3
	8	F	215	1.08	11:03	219.1	225.5	224.8	232.4	237.1
C-4	9	F	207	1.03	11:05	196.7	210.0	213.6	222.1	220.8
	10	F	212	1.05	11:06	199.5	210.1	210.8	220.0	227.6
D-l	1	M	230	1.15	11:07	234.0	240.4	258.4	244.4	286.3
Anatabine	2	M	248	1.25	11:21	242.6	254.0	267.9	255.1	268.1
1.5 mg/kg	3	M .	228	1.15	11:23	231.0	240.3	252.5	247.3	269.8
D-2	4	M	239	1.2	11:26	245.3	257.0	272.5	290.2	295.8
	5	M	227	1.15	11:28	227.8	237.3	250.1	262.0	270.5
D-3	6	F	216	1.08	11:08	213.9	212.0	220.0	226.2.	228.0
	7	F	206	1.03	11:09	204.0	206.2	210.3	216.8	217.5
	8	F	219	1.1	11:20	219.6	221.6	224.3	234.7	230.9
D-4	9	F	231	1.15	11:22	229.5	233.9	237.0	248.1	247.5
	10	F	206	1.03	11:23	206.1	208.6	211.4	221.8	218.9

128

Table 28B. Dosing Calculations and Body Weights, days 6-12 (anatabine)

2017201593 08 Mar 2017 day 6 day 7 day 8 day 9 day 10 day 11 day 12 B. W. B. W. B. W. B. W. B. W. B. W. B. W.

Group Rat M/F (g) (g) (g) (g) (g) (g) (g)

A-l	1 M	308	317.0	332.0	338.0	348.7	364.2	369.5
vehicle	2	M	283.7	300.0	310.0	315.0	327.2	342.6	342.6
5 mL/kg	3	M	287.9	300.0	314.0	317.0	332.0	349.1	354.1
A-2	4	M	268.8	273.5	286.0	286.9	296.1	312.7	316.7
	5	M	294.6	301.1	315.0	315.2	332.3	339.7	351.0
A-3	6	F	228.2	231.7	235.5	237.4	246.7	253.8	248.9
	7	F	. 235.3	236.8	260.8	249.8	252.8	261.3	256.3
	8	F	221.9	225.9	226.4	230.1	236.4	248.0	.240.9 '
A-4	9	F	216	217.4	218.0	224.8	228.5	237.1	238.8
	10	F	:. 232.4	232.5	233.0	238.3	241.9	256.7	256.9

129

2017201593 08 Mar 2017

Group	Rat	M/F	dav 6 B.W. (g)	dav 7 B.W. (g)	dav 8 B. W. (g)	dav 9 B.W. (g)	dav 10 B. W. (g)	dav 11 B.W. (g)	dav 12 B. W. (g)
C-l	1	M	282.4	294.5	300.0	307.6	317.2	334.6	327.6
Anatabine	2	M	287.6	302.9	310.6	319.8	331.9	339.8	325.4
0.75 mg/kg	3	M	268.2	281.8	293.4	298.8	307.9	329.4	322.5
C-2	4	M	268.2	274.1	284.2	296.2	303.8	316.1	324.8
	5	M	281.3	287.1	292.0	298.5	306.8	321.9	331.1
C-3	6	F	224.4	227.4	232.0	237.3	239.3	250.9	249.7
	7	F	217.8	223	220.0	226.3	232.2	239.1	233.5
	8	F	236.2	243.2	243.0	247.6	252.7	268.8.	259.6
C-4	9	' F	' 214.7	222	224.6	229.1	230.9	240.9	241.6
	10	F	221.4	219.3	227.4	23026	'233.5	244.1	247.9
D-l	1	M	279.1	292	300.0	302.1	317.1	333.0	341.1
Anatabine	2	M	296.9	305.6	319.0	323.1	331.8	359.6	360.3
1.5 mg/kg	3	M	280.4	288.2	298.4	303.8	318.3	335.4	327.9
D-2	4	M	302.3	316.7	326.0	337.3	347.0	369.5	380.4
	5	M	274.8	283.5	297.0	301.9	310.0	332.6	336.3
D-3	6	F	221.2	229	229.6	232.8	232.8	242.1	236.9
	7	F	204.6	208.1	216.4	218.7	229.0	233.0	227.4
	8	F	229.3	235.7	244.0	243.9	252.1	259.7	• **' 257.1
D-4	9	F	246	249.9	256.7	258.3	260.9	266.9	276.3
	10	F	216.9	224	225.0	225.8	234.5	243.5	240.9

130

2017201593 08 Mar 2017

Table 28C. Dosing Calculations and Body Weights, days 13-14 (anatabine) day 13 day 14

Group Rat M/F B. W. (g) B, W. (g)

A-l	1	M	379.1	389.0
vehicle	2	M	349	363.0
5 mL/kg	3	M	356	377.0
A-2	4	M	317	325.0
	5	M	356	371.9
A-3	6	F		265.2
	7	F		268.2
	8	F	245.6	252.7
A-4	9	F	235	245.8
	10	F	: 253	265.7
B-1	1	M	367	391.0
Anatabine	2	M	331	347.3
0.1 mg/kg	3	M	349	368.8
B-2	4	M	334.4	353.5
	5	M	360	373.8
B-3	6	F	271.8	272.7
	7	F	244	248.5
	8	F	241	243.9
B-4	9	F	242.1	255.6
	10	F	244	239.2

131 day 13 day 14

2017201593 08 Mar 2017

B. W.

B. W.

C-l	1	M	335.6	351.9
Anatabine	2	M	338	357.9
0.75 mg/kg	3	M	330	345.1
C-2	4	M	326	. 339
	5	M	326	335.9
C-3	6	/'////'F '//'Sir	.··' 248	251.5
	7		. ’ / 237.2	242.8
	8	F .	268	274.1
C-4	9	F	234	250.7
	10	/ ./ S/f -////Illi	i/' '/243iiii	250.6
D-l	1	M	339.8	359.8
Anatabine	2	M	370	389.7
1.5 mg/kg	3	M	340	' 357.1
, D-2	4 ’	M	379	399.9
	5	M	337	355
D-3	6	F	242.4	249.9
	7	F	223	224.4
	8	' F '	257	264.7
D-4	9 .	F	275.8	283.1
	10	F · : ·	240.3	248.8 '

132

2017201593 08 Mar 2017

133

2017201593 08 Mar 2017

134

2017201593 08 Mar 2017

135

Table 29A: Average Daily Food Consumption per Rat (grams)

Anatabine

2017201593 08 Mar 2017

A- Vehicle B - 0.1 mg/kg C - 0.75 mg/kg D - 1.5 mg/kg

Date	cage#	# of rats	Male	Female	Male	Female	Male	Female	Male	Female
day 1	1	3	25.1	14.1	25.8	16.4	23.9	17.1	22.7	16.1
2	2	21.1	15.4	28.0	14.4	19.2	17.1	21.4	13.6
day 2	.1	3	27.4	16.3	33.8	20.2	26.3	22.5	24.2	20.5
2	2	24.5	20.4	29.1	20.4	29.5	11.3	30.2	19.8
day 3	1	3	25.4	16.8	24.8	19.4	28.1	21.0	30.1	19.6
2	2	25.3	20.3	22.0	18.2	26.3	17.4	31.2	20.9
day 4	1	3	26.8	17.6	34.1	23.2	28.3	21.0	22.1	20.8
2	2	26.4	20.2	28.4	18.0	29.8	18.7	30.0	21.6
day 5	1	3	28.5	17.2	30.6	22.4	24.4	19.1	30.0	18.5
2	2	28.3	22.4	28.6	21.4	27.0	21.7	29.0	18.5
day 6	1	3	26.7	20.6	33.6	26.8	25.6	18.8	33.6	14.2
2	2	26.0	19.2	28.0	18.2	29.5	, 16.4	35.4	18.4
day 7	1	3	30.1	21.6	35.7	21.7	29.0	24.0	34.1	21.2
2	2	27.9	20.5	31.8	22.4	31.5	17.4	33.5	28.8
day 8	1	3	33.2	23.7	33.2	26.5	31.3	23.2	31.7	22.7
2	2	31.5	23.8	31.9	21.3	32.6	22.8	34.4	19.8
day 9	1	3	29.1	24.1	35.5	21.1	29.1	23.7	33.9	21.7
2	. 2	27.7	24.9	36.8	23.6	30.3	23.0	34.5	23.1
day 10	1	3	30.4	22.3	34.7	20.4	28.7	20.0	33.5	21.7
2	2	29.4	20.5	26.7	16.9	28.0	21.0	32.4	21.7
day 11	1	3	28.0	19.9	29.1	19.0	24.4	20.5	28.3	15.6
2	2	24.4	22.9	30.6	19.1	26.7	18.6	29.9	19.0
day 12	1	3	27.4	20.3	31.6	27.9	21.7	16.6	30.1	18.4
2	2	25.6	18.8	34.9	16.7	31.0	20.4	33.1	20.8
day 13	1	3	33.6	22.4	39.9	29.6	33.9	22.0	36.8	19.6
2	2	30.8	20.9	27.1	19.3	27.2	17.7	34.7	20.5
day 14	1	3	28.7	20.9	32.3	18.8	27.8	17.6	29.9	17.1
2	2	26.4	19.6	28.8	19.1	26.4	20.7	31.4	19.2

136

Table 29B: Average Daily Food Consumption per Rat (grams)

2017201593 08 Mar 2017

E- Nicotine 0.75 mg/kg

Males Female

Date	cage#	# of rats	grams	# of rats	grams
day 1	1	3	20.8	1	23.7
2	1	19.3	2	14.8
day 2	1	3	23.5	1	24.9
2	1	26.2	2	18.4
day 3	1	3	26.6	1	22.2
2	1	26.2	2	22.3
day 4	1	3	26.2	1	19.4
2	1	23.7	2	15.8
day 5	1	3	25.1	1	18.7
2	1	21.9	2	16.2
day 6	1	3	23.8	1	20.0
2	1	25.9	2	17.2
day 7	1	3	26.5	1	19.4
2	1	27.9	2	21.7
day 8	1	3	24.0	1	20.1
2	1	28.2	2	20.6
day 9	1	3	27.3	1	24.4
2	1	26.1	2	21.5
day 10	1	3	29.6	1	22.6
2	1	30.9	2	20.4
day 11	1	3	30.1	1	20.4
2	1	29.1	2	20.3
day 12	1	3	26.5	1	22.5
2	1	23.4	2	21.7
day 13	1	3	31.9	1	23.7
2	1	28.4	2	21.2
day 14	1	3	32.7	1	24.4
2	1	26.4	2	19.6

137

Table 30. Hematology/Coagulation Parameters: Normal Ranges in the Rat

2017201593 08 Mar 2017

	Unit of Measure	Range
WBC	x 10J/gL	3.0- 17.0
RBC	x 106/gL	5-10
HGB	g/dL	11-19
HCT	%	35-57
MCV	fL	46-65
MCH	pg	18-23
MCHC	g/dL	31-40
RETICULOCYTE COUNT	%	0-25
NEUTROPHIL SEG	%	7-15
LYMPHOCYTE	%	77-89
MONOCYTE	%	0-5
EOSINOPHIL	%	0-4
BASOPHIL	%	0-1
PLATELET COUNT	x107gL	200- 1500
aPTT	sec	13.2-22.4
PT	sec	11.0-15.6

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2017201593 08 Mar 2017

Table 32. Clinical Chemistry Parameters: Normal Ranges in the Rat

	Unit of Measure	Range
ALKALINE PHOSPHATASE	IU/L	160- 500
ALT (SGPT)	IU/L	35-80
AST (SGOT)	IU/L	33-53
GLOBULIN	g/dL	1.4-5.0
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TOTAL PROTEIN	g/dL	4.5-8.4
TOTAL BILIRUBIN	mg/dL	0 - 0.64
BLOOD UREA NITROGEN (BUN)	mg/dL	11-23
CREATININE	mg/dL	0.4-3.8
CHOLESTEROL	mg/dL	35-75
GLUCOSE	mg/dL	80 - 300
CALCIUM	mg/dL	9.1 - 15.1
PHOSPHORUS	mg/dL	4.7-16.0
CHLORIDE	mEq/L	79-111
POTASSIUM	mEq/L	3.6-9.2
SODIUM	mEq/L	142-154

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2017201593 08 Mar 2017

Clinical Chemistry Parameters

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147

2017201593 08 Mar 2017

Clinical Chemistry Parameters

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151

2017201593 08 Mar 2017

Table 34. Coagulation Parameters

Coagulation Parameters

Animal ID	Sex	Anatabine ACTIVE PARTIAL THROMBOPLASTIN TIME PROTHROMBIN TIME
Dose (mg/kg)	seconds	seconds
Al	M	0	33.5	13.3
A2	M	0	31.7	13.3
A3	M	0	29.6	12.5
A4	M	0	30.4	. 12.3
A5	M	0	34.9	13.1
A6	F	0	. 36.7	12.8
A7	F	0	36.2	12.7 .
A8	F	0	40.6	13.2
A9	F	0	34.5	12.5
A10	F	0	29.2	12.5
Bl	M	0.1	34.9	13.6
B2	M	0.1	36.4	13.4
B3	M	0.1	29.6	12.5
B4	M	0.1	28.9	12.3
B5	M	0.1	32	12.1
B6	F	0.1	31.1	12.3
B7	F	0.1	34.2	11.3
B8	F	0.1	26	12.2
B9	F	0.1	26.7	12.3
BIO	F	0.1	28.9	12.3

152

Coagulation Parameters

2017201593 08 Mar 2017

Animal ID	Sex	Anatabine ACTIVE PARTIAL THROMBOPLASTIN TIME PROTHROMBIN TIME
Dose (mg/kg)	seconds	seconds
Cl	M	0.75	39.7	12.4
C2	M,	0.75	43.9	12.1
C3	. M	0.75	27.9	12.1
C4	M	0.75	50.4	17.9
C5	M	0.75	31.7	12.1
C6	F	0.75	31.4	11.4
C7	F	0.75	32.5	12.1
C8	F	0.75	31.5	11.6
C9	F	0.75	32.7	11.4
CIO	F	0.75	38.2	23.8
DI	M	1.5	34.1	13.8
D2	M	1.5	33.8	13.1
D3	M	1.5	28.3	12.9
D4	M	1.5	27.9	12.9
D5	M	1.5	29.1	13.5
D6	F	1.5	37.2	13.2
D7	F	1.5	31.7	13.2
D8	F	1.5	29.3	13
D9	F	1.5	32.3	13.6
DIO	F	1.5	32	13.3

153

Coagulation Parameters

2017201593 08 Mar 2017

Animal ID	Sex	Anatabine Dose (mg/kg)	ACTIVE PARTIAL THROMBOPLASTIN TIME	PROTHROMBIN TIME
seconds	seconds
El	M	0.751	15	13.8
E2	M	0.75'	16.3	13.2
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E5	M	0.75'	16.2	13
E6	F	0.75'	18.7	13.9
E9	F	0.751	15	14.2
				UNABLE TO OBTAIN
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E10	F	0.751	CLOTS	FIBRIN CLOTS

1. Dose is 0.75 mg/kg nicotine

154

2017201593 08 Mar 2017

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157

2017201593 08 Mar 2017

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0.41

r- - - .'

3.46

2.04

Cass

| Tissue

Thymus

Heart

Lungs

Thyroid/para thyroid

Liver

Adrenals

Kidneys

O JU. · 'Έχ. 00

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

<*>

weights

08Ό

ΓΟΟ

2.16

3 u

15.67

5Λ -.3 Q

r* t© cn

Wi

0.47

0.56

3.31

: O c4

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

<u Λ ΤΛ ' 2 .

Testes

Brain

Pituitary

Marrow

| weights 1

08Ό

OO Γη

1.67

Cass

17.01

& o

3.51

to 00 \ o

0.56

3.33

2.04

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

I weightsl

08Ό

1.65

2.02

Cass

17.19

Cass

3.86

i 1.12

0.53

0.31

3.40

Γ4 OO

Cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

158

2017201593 08 Mar 2017

Ο

weights |

0.66

1.22

1.56

Cass

11.49

V)· Λ u

2.42

Ι9Ό

1.02

; 19'0

1.76

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

OS

weights |

0.55

1.14

1.41

C/i ' ω ee.··. o

9.36

5Λ ω U

1.75 i

0.50

0.44

OO OO

2.12

cass

1 Tissue I

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

QO

weights |

0.78

^F cn p—H

1.36

C/5 C/3 co u

6101

vi ο

2.14

0.56

0.90

111'835/

1.85

cass

-?

I Tissue I

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights |

0.79

1.31

09Ί

Cass

11.24

Cass

2-72

0.68

0.41

CN rF

2.06

cass

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals ____________________ 1

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights |

99Ό

1.33

1.67

Cass

12.40

Cass

2.66

0.72

1.33

0.76

1.87

cass

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

159

Γο

ΓΗ re

S ο

weights |

18Ό

1.85

1.83

Cass

17.38

Cass

3.99

v© ro

0.35

0.48

3.29

1.88

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate i

Testes

.£ ' 're «

Pituitary

Marrow

□k ο

CM

ΓΟ ΓΗ

weights |

0.71

09T

Ok

Cass

16.32

Cass

3.27

0.93

0.50

WY o

3.35

CM

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ I parathyroid

Liver

Adrenals ~

Kidneys

c 4) jy> • Ή.'

Small intestine

Prostate

Testes

.£ ’re . CQ ,

Pituitary

Marrow

weights |

0.76

Ok

2.08

Cass

86'91

Cass

3.70

0.82

88Ό

©

3.14

2.24

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ parathyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate.

Testes

Brain

Pituitary

Marrow

weights |

0.74

1.59

1.77

Cass

15.45

Cass

3.33

990

960

0.49

3.64

2.16

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ parathyroid |

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights |

0.74

Γη

2.31

U

17.92

Cass

3.55

16Ό

Ι9Ό

0.72

3.69

/ 2.35

cass

-7

Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

160 cο cd

S s

o

10

I weights |

0.73

1.25

1.59

Cass

9.67

r CO : CO ¢3

2.25

0.65

0.63

pr——- 0.70

1.89

cass

| Tissue

Thymus

Heart ι

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine |

Ovaries/ uterus

Brain

Pituitary

Marrow

m □s m o cd ro cd

weights |

0.71

1.05

1.52

Cass

12.94

co co CQ <_>

2.14

' O

0.49

Cd re,

1.86

CO . S3. Q

| Tissue

Thymus

. Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus ι

Brain

Pituitary

Marrow

weights |

0.49

un o ’©

Cass

9.94

CO CO C5 o

2.02

o

0.55

0.84

1.75

cass

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

| weights |

0.59

1.12

1.39

Cass

60ΌΙ

// co 3 O

2.02

Cd © · o

0.71

68T

co Q

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

| weights |

890

1.29

1.42

Cass

11.54

Cass

2.46

rd

0.87 i

96Ό

161

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

161

Γο CN fe s ο

•n

weights |

0.47 1

1.29

1.58

Cass

14.94

-- Cass

2.89

0:64

0.47

0.45

2.70

CN O CN

cass

I Tissue |

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

£ o fc s

m □k o CN ΓO CN

’fr

weights |

69Ό

1.39

1.95

Cass

14.01

Cass

3.16

0.93

0.41

<© . ra o

3.28

2.00

cass

I Tissue 1

Thymus

Heart

I Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

1 Pituitary

Marrow

weights |

0.67

x© in

1.72

GO 531 u

14.70

Cass

3.49

Ok Γ**» o

0.56

in O'

3.03

2.12

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

| weights

0.52

1.38

1.97

Cass

13.91

Cass

2.77

x© o

-ra

0.49

3.84

2.19

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights |

1.02

1.52

1.86

Cass

14.92

Cass

3.45

0.71

0.54

0.49

3.19

2.01

cass

I Tissue

Thymus

Heart

Lungs

Thyroid/ para , thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

162

Γο

CN s

ο

o

weights |

0.54

66Ό

1.49

(Z) P3 U

6Γ01

Cass

2.36

0.49

1.34

3.65

OO

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

□8 in o

CN

Γo CN

Cs

weights |

0.52

1.03

1.30

Cass

11.20

Cass

2.18

0.51

0.47

cn CM

2.06

cass

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

. Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights

0.63

1.23

1.62

co u

11.37

Cass

2.68

0.65

0.85

CM ©

1.73

ΐΛ CO C3 Q

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

G <υ 5-

Small intestine

7/5 FA .2'5 Έ - w c>5 < /3 Ο X

Brain

Pituitary

Marrow

weights |

0.53

1.21 /

1.39

Cass

8.65

Cass

2.20

0.65

ΓΓη c>

1.50

1.93

cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights |

0.56

60T

1.46

Cass

10.71

Cass

2.08

0.70

0.72 1

0.81 1 / ·

66 1

cass

-?

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

163

Γο

CN

S

Σ ο

| weights 1

0.59

1.47

06'1

Cass

14.26

Cass

3.43

0.92

99Ό

0.45

3.23

2.08

cass

1 Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

m □s χ ο CN

ΓΟ CN

Tf

weights |

98Ό

X

2.02

Cass

18.27

Cass

3.79

r* o · o

1.00

C\ Ν’ O

3.41

2.00

cass

| Tissue |

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights |

0.54

1.47

1.93

Cass

17.35

Cass

3.02

0.86

0.56

3.51

2.02

cass

| Tissue |

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

. Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights |

0.77

1.53

2.06

Cass

17.03

c/i a o

3.54

0.83

0.77

X Tf o

3.28

1.81

cass

I Tissue |

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

| weights |

0.83

1.50

1.77

Cass

15.86

Cass

3.48

0.71

0.43

0.50

2.78

1.75

cass

7-

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

164 ο

CM re s

o

o

weights |

0.51

86Ό

1.59

Cass

9.21

Cass

1.97

0.51

0.77

0.78

Ok

cass

~7

I Tissue

Thymus

Heart

Lungs

Thyroid/ parathyroid

Liver

Adrenals

Kidneys

- . Spleen

Small intestine

Ovaries/ | uterus ___I

Brain

Pituitary

Marrow

□k o

CM

Γo CM

weights |

Ι9Ό

0.97

1.60 1

CA-

11.05

Cass

2.64

0.68

0.56

1.34

2.08

cass

~7

I Tissue

Thymus

Heart

Lungs

Thyroid/ parathyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ . uterus

Brain

Pituitary i

Marrow

weights |

0.52

o

1.37

CO o

11.46

Cass

2.52

re·' ,-o

1.07

0.98

Ok

cass

7

Tissue 1

Thymus

Heart :

Lungs

Thyroid/ parathyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus i

Brain

Pituitary

Marrow

weights |

0.63

0.88

1.46

Cass

8.83

CO a o

2.08

0.65

09Ό

1.20

Ok

CO co re Q

~7

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid i

Liver

Adrenals i

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights |

0.59

CM

1.29

Cass

9.65

Cass

2.17

0.53

0.49

0.84

1.90

cass

~~7

1 Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

165

Γο CN fe s ο

m □k o CN ΓO CN

weights |

0.76

1.42

1.66

Cass

14.20

Cass

3.01

98Ό

0.40

00 CN o

2.92

1.94

Cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights I

0.56

CN

1.62

Cass

12.06

Cass

2.87

0.78

0.53

0.45

CN O en

1.84

Cass

1 Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights |

0.46

1.17

1.49

Cass

12.52

Cass

3.04

0.62

0.56

0.28

3.13

06T

Cass

I Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

weights |

0.54

1.20

1.84

Cass

11.97

Cass

3.19

0.81

0.71

0.41

3.05

2.06

Cass

| Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals i

Kidneys

Spleen

Small intestine

Prostate

Testes

Brain

Pituitary

Marrow

166

Γο

CN s

ο m

□k o

CN ro CN

weights |

0.48

0.83

1.28

Cass

9.18

Cass

2.29

0.46

0.28

CN cn

1.87

Cass

Tissue

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights |

0.48

90T

00

Cass 1

d

Cass

2.29

0.76

0.40

0.92

1.93

Cass

| Tissue |

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

weights |

0.54

60T

1.20

Cass

1 9.48

Cass

2.34

0.46

0.41

1.59

1.77

Cass

I Tissue |

Thymus

Heart

Lungs

Thyroid/ para thyroid

Liver

Adrenals

Kidneys

Spleen

Small intestine

Ovaries/ uterus

Brain

Pituitary

Marrow

167

2017201593 08 Mar 2017

Table 37. Dosing solutions

Test compound	Percentage content of anatabine	Dose level (mg/kg)	Test compound concentration (total) (mg/mL)	Test compound concentration (base) (mg/mL)	Injection volume (mL/kg)
Anatabine	5.18	0.20	0.772	0.04	5
Anatabine	5.18	2.0	7.72	0.4	5

Table 38. Dosing

Test compound	Route	Sex	Concentration mg/kg/ dose	# of animals	Dosing times (minutes)
Anatabine	p.o.	M	0.2	4	0, 240,480
F	0.2	4
M	2.0	4
F	2.0	4

Table 39. Blood Collection Times

Test compound	Route	Sex	Concentration mg/kg/ dose	# of animals	Blood collection (minutes)
Anatabine	p.o.	M	0.2	4	30, 60, 235 (pre-dose), 270, 300, 475 (predose), 540, 600, 720, 1440
F	0.2	4
M	2.0	4
F	2.0	4

168

2017201593 08 Mar 2017

Table 40. Calibration Curve Concentrations

nominal concentration (n.M)	stock concentration (μΜ)
5000	250 ‘
1667	83.3
555.5	27.8
185.2	9.3
61.7	3.1
20.6	1.0
6.9	0.34
2.3	0.11
0.76	0.038
0.25	0.013

Table 41. LC/MS/MS ionization conditions and identity of parent and product ions.

Compound	MW	Polarization	Precursor m/z	Product m/z	Collision energy (V)
Anatabine	160.2	Positive	161.1	115.1	28
(JJ,S)-Antabine-2,4,5,6-d4	164.24	Positive	165.1	148.1	20

Table 42. Limits of Detection and Calibration Curves

Sample	Limit of Detection (LOD) (ng/mL)	Lower Limit of Quantitation (LLQ) (ng/mL)	Upper Limit of Quantitation (ULQ) (ng/mL)
Anatabine in rat plasma	0.37	1.1	>801

169

Table 43. Dosing Solution Analysis

2017201593 08 Mar 2017

Compound	Dose (mg/kg)	Expected Concentration (mg/mL)	Actual Concentration (mg/mL)	Actual Concentration relative to Expected (%)
anatabine	0.2	0.04	0.025	63%
anatabine	2.0	0.4	0.335	84%

170

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Anatabine (6.0 mg/kg)	cu	0.255	8160	0.430	899Ό	8990
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Mean	3257	1.76	5.07	3.18	0.73
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Mean	3496	1.79	4.53	2.97	0.52
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175

2017201593 08 Mar 2017

Table 50. Measured Concentrations of Anatabine in Rat Plasma Samples at Each Time Point

Dose (mg/kg)	Animal ID	Sex	Time Point (min)	Anatabine Concentration (ng/mL)
0.2	IA	male	30	34
60	26
235	11
270 '	35
300	22
475	9
540	4
600	<LOQ
720	<LOQ
1440	<LOQ
IB	male	30	<LOQ
60	32
235	<LOQ
270	31
300	26
475	<LOQ
540	28
600	17
720	5
1440	<LOQ
IC	male	30	29
60	34
235	13
270	20
300	29
475	18
540	41
600	35
720	12
1440	<LOQ
ID	male	30	37
60	24
235	9

176

2017201593 08 Mar 2017

			270	26
300	22
475	19
540	43
600	34
720	19
1440	<LOQ
2A	female	30	34
60	29
235	4
270	14
300	18
475	7
540	24
600	12
720	18
1440	<LOQ
2B	female	30	34
60	33
235	18
270	11
300	31
475	7
540	44
600	18
720	15
1440	<LOQ
2C	female	30	42
60	47
235	6
270	17
300	25
475	6
540	29
600	20
720	7

177

2017201593 08 Mar 2017

			1440	<LOQ
2D	female	30	38
60	25
235	13
270	51
300	44
475	10
540	29
600	45
720	15
1440	<LOQ
2	3A	male	30	298
60	153
235	84
270	131
300	312
475	82
540	223
600	269
720	133
1440	12
3B	male	30	288
60	106
235	46
270	236
300	232
475	79
540	214
600	173
720	401
1440	<LOQ
3C	male	30	269
60	272
235	63
270	364
JOO	116

178

2017201593 08 Mar 2017

			475	97
540	290
600	130
720	137
1440	42
3D	male	30	116
60	116
235	105
270	309
300	202
475	114
540	173
600	150
720	71
1440	36
4A	female	30	245
60	81
235	75
270	237
300	216
475	144
540	231
600	197
720	186
1440	42
4B	female	30	78
60	95
235	36
270	324
300	97
475	219
540	314
600	207
720	165
1440	8
4C	female	30	218

179

2017201593 08 Mar 2017

			60	127
235	23
270	273
300	191
475	178
540	369
600	480
720	244
1440	36
4D	female	30	98
60 .	165
235	72
270	350
300	414
475	179
540	474
600	288
720	217
1440	<LOQ

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181

2017201593 08 Mar 2017

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182

2017201593 08 Mar 2017

EXAMPLE 7

Effect of S-(-)-anatabine on TNFa-induced NFkB activity in vitro [217] The effect of S-(-)-anatabine on TNFa-induced NFkB activity in vitro was determined as described in Example 1. NFkB activity was stimulated with 20ng/ml of TNFa, then varying doses of a racemic mixture of anatabine or S(-)-anatabine were applied to the challenged cells. The data were plotted as a percentage of the TNFa-induced NFkB activity and are shown in FIG. 24. In this assay the IC50 for the racemic mixture of anatabine is approximately 600pg/ml, whereas the IC50 for the (S)-enantiomer is approximately 330pg/mI.

183

2017201593 08 Mar 2017

EXAMPLE 8

Step 1. Preparation of Formula I [218] 3-aminomethylpyridine was added to neat benzophenoneimine (1 eq). The reaction was allowed to proceed for 6 hours at 50 °C providing Formula I as the product.

EXAMPLE 9

Steps 2i to 2iii: Conversion of Formula I to Anatabine using Potassium Tert Butoxide (K'OBu) |219] Step 2i: To Formula I of Example 1 was added K'OBu (1.5 eq) in THF at -78 °C to -45 °C and the reaction was incubated for 30 min. Cis-l,4-dichloro-2butene in 3 vol of THF at -78°C was added, and the temperature was allowed to warm to -45°C. The reaction was allowed to proceed at -45°C for 1-2 hours, providing Formula II. Optionally, the starting material may be added to the K'OBu.

[220] Step 2ii: 10% aqueous hydrochloric acid was added to the solution containing Formula II for 10-20 minutes to provide Formula III.

[221] Step 2iii: The Formula III solution was basified by adding K2CO3 then treated with a 40% aqueous KOH solution to provide anatabine.

[222] Results from different batches are show in Table 52.

Table 52

Batch No. and amount	Reaction Conditions	Yield	Comments
A (2g)	i) K‘OBu (1.1 eq), THF,78°C to -45°C for 30 min ii) cis-1,4-dichloro-2butene -45°C for 1 -2h iii) 10% aq HCI, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH	0.7g (crude)	SM was added to K'OBu. 64.52 % of Step 2i product Anatabine 65.12% pure by HPLC
B (2g)	i) K'OBu (1.5eq), THF,- 78°C to -45°C for 30 min	0.68g (crude)	Note that 1.5 eq of K'OBu was used.

184

2017201593 08 Mar 2017

	ii) cis-l,4-dichloro-2butene -45°C for l-2h iii) 10% aq HC1, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH		70.32% of Step 2i product Anatabine 67.06% pure by HPLC
c (25g)	i) K‘OBu (1.1 eq), THF,78°C to -45°C for 30 min i i)c is-1,4-dichloro-2butene -45°C for l-2h iii) 10% aq HC1, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH	8.5g (crude)	50 % of Step 2i product Anatabine 57.7% pure by HPLC

EXAMPLE 10

Steps 2i to 2iii alternate route: Conversion of Formula I to Anatabine using LDA [223] Formula I was prepared according to Example 1. LDA was added to Formula I at -10 to 0 °C. Cis-l,4-dichloro-2-butene was added at -78 °C to -45 °C. Steps 2ii and 2iii were performed according to Example 9.

185

2017201593 08 Mar 2017

Table 53

Batch No. and amount	Reaction Conditions	Yield	Comments
D (25g)	i) LDA (1.2eq), THF, -30 °C to 0 °C for 30 min ii) cis-l,4-dichloro-2butene -78 °C to -45 °C for l-2h iii) 10% aq HCI, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH	6.9g (crude)	71 % conversion in IPC. 23.14% SM was seen in Step 2i. 72.64% purity by HPLC.
E (5g)	i) LDA (1.5eq) -30 °C to 0 °C for 30 min ii) cis-1,4-dichloro-2butene -78 °C to -45°C for l-2h iii) 10% aq HCI, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH		80 % conversion in IPC Anatabine 70.6% pure by HPLC
F (25g)	i) LDA (2.0eq), -30 °C to 0 °C for 30 min ii) cis-l,4-dichloro-2butene -78 °C to -45°C for l-2h iii) 10% aq HCI, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH		67 % conversion in IPC Anatabine 76.1% pure by HPLC
G (25g)	i) LDA (1.5eq), -30 °C to 0 °C for 30 min ii) cis-l,4-dichloro-2butene -78 °C to -45°C for l-2h iii) 10% aq HCI, 10-20 min, ether wash. iv) K2CO3, 40% aqueous KOH		Anatabine 73.7% pure by HPLC

EXAMPLE 10A

Recovery/Purification of Anatabine Using MTBE and Distillation [224] The product of Example 9, Step 2iii was extracted with methyl t-butyl ether (MTBE), followed by distillation of the solvent and product distillation using a

186

2017201593 08 Mar 2017 glass distillation assembly. Optionally, for scale-up, wiped film or thin film evaporation may be used. Yield of step 2 was 40%; overall yield was 26%.

COMPARATIVE EXAMPLE 10A

Recovery/Purification of Anatabine Using Chloroform Column Chromatography [225] The product of Step 2iii was treated with chloroform extraction as described in Deo et al. Chloroform extraction resulted in an anatabine yield of 10%.

EXAMPLE 11

Stability analysis of Formula I [226] Several batches of Formula I produced by reacting benzophenoneimine with aminomethylpyridine in the absence of benzene were stored refrigerated and also at'room temperature. Testing by HPLC shows that purity was constant when refrigerated and only deviated 1-2% at room temperature.

Table 54

Days	Purity (%) (stored at 2-8°C)	Purity (%) (stored at 25-28°C)
1	88.61	88.93
2	88.83	85.49
3	88.30	88.02
6	88.83	86.62
12	88.75	87.47
18	88.46	87.25

EXAMPLE 12

Stability Analysis of Anatabine Base With and Without BHT as a Preservative [227] Anatabine base stability was analyzed with and without BHT (3,5-di-tertbutyl-4-hydroxytoluene). The initial purity of BHT-free anatabine base was 94.95%. The initial purity of anatabine base with BHT was 94.87%. Anatabine stability, with and without BHT is reported in Table 55.

187

2017201593 08 Mar 2017

Table 55

Day	Storage Temperature	Purity (%) (with BHT)	Purity (%) (without BHT)
1	25-28 °C	93.27	89.63
2-8 °C	93.26	94.44
-20 °C	94.26	93.90
2	25-28 °C	92.73	87.59
2-8 °C	94.83	94.05
-20 °C	94.73	94.62
3	25-28 °C	87.44	80.74
2-8 °C	94.26	91.65
-20 °C	94.62	93.87
12	25-28 °C	85.10	79.59
2-8 °C	94.18	90.12
-20 °C	94.74	94.01

EXAMPLE 13

Preparation of Anatabine Salts [228] To a solution of anatabine (4.0 g, 24.9 mmol) in acetone (20 ml), L-(+)-tartaric acid (3.37, 22.4 mmol) was added at room temperature. The reaction mass was warmed to 50 °C for 16 hours. The supernatant was decanted and the solid was triturated with diethyl ether (20 ml), filtered and dried under vacuum.

Table 56

Solvent Used	Anatabine Tartrate (% Purity by HPLC)
Isopropyl alcohol	> 94%
Acetone	> 99%

[229] To a solution of anatabine (0.47g, 2.9mmol) in acetone (3 ml), citric acid (0.5g, 2.6mmol) was added at room temperature under a nitrogen atmosphere.

The reaction mass was warmed to 50 °C for 16 hours. The supernatant was

188 decanted and the solid was triturated with diethyl ether (20 ml), filtered and dried under vacuum.

2017201593 08 Mar 2017

Table 57

Solvent Used	Anatabine Citrate (% Purity by HPLC)
Acetone	> 97%

[230] While particular embodiments have been described and illustrated, it should be understood that the invention is not limited thereto since modifications may be made by persons skilled in the art. The present application contemplates any and all modifications that fall within the spirit and scope of the underlying invention disclosed and claimed herein.

Claims (13)

1. CLAIMS:

   1. Use of an isolated form of anatabine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing a symptom of thyroiditis.
2. 2. Use of an isolated form of anatabine or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing a risk of thyroiditis.
3. 3. Use according to claim 1 or 2, wherein anatabine is present as an isolated form of S-(-)anatabine or a pharmaceutically acceptable salt thereof.
4. 4. Use according to any one of the preceding claims, wherein anatabine is present as an isolated form of R-(+)-anatabine or a pharmaceutically acceptable salt thereof.
5. 5. The use according to any one of the preceding claims, wherein the pharmaceutically acceptable salt is anatabine citrate.
6. 6. A method of reducing a symptom in an individual of thyroiditis, comprising administering to the individual a pharmaceutical composition comprising a therapeutically effective dose of an isolated form of anatabine or a pharmaceutically acceptable salt thereof.
7. 7. A method of reducing a risk in an individual of developing thyroiditis, comprising administering to the individual a composition comprising a therapeutically effective dose of an isolated form of anatabine or a pharmaceutically acceptable salt thereof.
8. 8. The method of claim 6 or claim 7 wherein the composition comprises a pharmaceutically acceptable salt of anatabine.
9. 9. The method of claim 6 or claim 7 wherein the dose is in an extended release formulation.
10. 10. The method of claim 6 or claim 7 wherein the dose is from about 0.1 to about 1.5 mg/kg body weight.
11. 11. The method according to any one of claims 6 to 10, wherein anatabine is present as an isolated form of S-(-)-anatabine or a pharmaceutically acceptable salt thereof.
12. 12. The method according to any one of claims 6 to 10, wherein anatabine is present as an isolated form of R-(+)-anatabine or a pharmaceutically acceptable salt thereof.
13. 13. The method according to any one of claims 6 to 12, wherein the pharmaceutically acceptable salt is anatabine citrate.